Targeting Ligands For Therapeutic Compounds

ABSTRACT

Described are novel targeting ligands that may be linked to compounds, such therapeutic compounds, that are useful in directing the compounds to the target in vivo. The targeting ligands disclosed herein can serve to target expression-inhibiting oligomeric compounds, such as RNAi agents, to liver cells to modulate gene expression. The targeting ligands disclosed herein, when conjugated to an expression-inhibiting oligomeric compound, may be used in a variety of applications, including use in therapeutic, diagnostic, target validation, and genomic discovery applications. Compositions including the targeting ligands disclosed herein when linked to expression-inhibiting oligomeric compounds are capable of mediating expression of target nucleic acid sequences in liver cells, such as hepatocytes, which may be useful in the treatment of diseases or conditions that respond to inhibition of gene expression or activity in a cell, tissue, or organism.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/542,423, filed on Mar. 7, 2017, which claims priority to U.S.Provisional Patent Application Ser. No. 62/304,652, filed on Mar. 7,2016, and U.S. Provisional Patent Application Ser. No. 62/370,754, filedon Aug. 4, 2016, and U.S. Provisional Patent Application Ser. No.62/426,916, filed on Nov. 28, 2016, the contents of each of which areincorporated herein by reference in their entirety.

BACKGROUND

Many compounds need to be delivered to a specific location (for example,to desired cell(s)) to have a therapeutic effect or to be useful fordiagnostic purposes. This is frequently the case when attempting todeliver a therapeutic compound in vivo. Further, being able toefficiently deliver a compound to a specific location can limit orpotentially eliminate unintended consequences (such as off-targeteffects) that may be caused by administration of the compound. Onemethod to facilitate delivery of a compound, such as a therapeuticcompound, to a desired location in vivo, is by linking or attaching thecompound to a targeting ligand.

One class of therapeutic compounds that can be targeted using targetingligands are oligomeric compounds. Oligomeric compounds that includenucleotide sequences at least partially complementary to a targetnucleic acid have been shown to alter the function and activity of thetarget both in vitro and in vivo. When delivered to a cell containing atarget nucleic acid (such as mRNA), oligomeric compounds have been shownto modulate the expression of the target resulting in alteredtranscription or translation of the target nucleic acid. In certaininstances, the oligomeric compound can reduce the expression of the geneby inhibiting the nucleic acid target and/or triggering the degradationof the target nucleic acid.

If the target nucleic acid is mRNA, one mechanism by which anexpression-inhibiting oligomeric compound can modulate the expression ofthe mRNA target is through RNA interference. RNA interference is abiological process by which RNA or RNA-like molecules (such aschemically modified RNA molecules) are able to silence gene expressionthrough degradation. The process of post-transcriptional gene silencingis thought to be an evolutionarily-conserved cellular defense mechanismused to prevent the expression of foreign genes.

Synthetic RNA and RNA-like molecules have been shown to elicit RNAinterference in vivo. For example, Elbashir et al. (Nature 2000, 411,494-98) describes RNAi induced by introduction of duplexes of synthetic21-nucleotide RNA molecules in cultured mammalian cells. The types ofsynthetic RNA or RNA-like molecules that can trigger the RNAi responsemechanism may be comprised of modified nucleotides and/or one or morenon-phosphodiester linkages.

Additionally, single-stranded RNA and RNA-like molecules, which can alsoinclude modified nucleotides and have one or more non-phosphodiesterlinkages, can also alter the expression of a target nucleic acid, suchas a target mRNA.

SUMMARY

Disclosed herein are targeting ligands that can enhance the delivery oftherapeutic compounds to a specific target site, e.g., a specific organor tissue, within a subject such as a human patient or animal. In someembodiments, the targeting ligands described herein can enhance thetargeted delivery of expression-inhibiting oligomeric compounds. In someembodiments, the targeting ligands can enhance the delivery ofexpression-inhibiting oligomeric compounds to the liver.

The targeting ligands disclosed herein include or consist of one or moretargeting moieties, one or more tethers, one or more branch pointgroups, and one or more linkers.

Disclosed herein are targeting ligands that include, consist of, orconsist essentially of the general structure of Formula A of FIG. 20,wherein n is an integer from 1 to 4 (e.g., 1, 2, 3, or 4).

In some embodiments, the targeting ligands disclosed herein include,consist of, or consist essentially of the structure of Formula B:

wherein n is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20); X is O, S, or NH; andTargeting Moiety is selected from the group consisting of:N-acetyl-galactosamine, galactose, galactosamine,N-formyl-galactosamine, N-propionyl-galactosamine,N-n-butanoylgalactosamine, and N-iso-butanoylgalactosamine.

In some embodiments, the targeting ligands disclosed herein include,consist of, or consist essentially of, the following structure:

wherein n is an integer from 1 to 20 (Structure 1).

In some embodiments, the targeting ligands disclosed include, consistof, or consist essentially of, the structure selected from:

The targeting ligands disclosed herein include one or more targetingmoieties. In some embodiments, the targeting ligands disclosed hereininclude N-acetyl-galactosamine as the targeting moiety.

The targeting ligands disclosed herein can be linked, directly orindirectly, to a compound, such as a therapeutic compound, e.g., anexpression-inhibiting oligomeric compound, for example, to the 3′ or 5′terminal end of the expression-inhibiting oligomeric compound. In someembodiments, the expression-inhibiting oligomeric compound includes oneor more modified nucleotides. In some embodiments, theexpression-inhibiting oligomeric compound is an RNAi agent, such as adouble-stranded RNAi agent. In some embodiments, the targeting ligandsdisclosed herein are linked to the 5′ terminal end of the sense strandof a double-stranded RNAi agent. In some embodiments, the targetingligands disclosed herein are linked to the RNAi agent via a phosphate,phosphorothioate, or phosphonate group at the 5′ terminal end of thesense strand of a double-stranded RNAi agent.

Disclosed herein are compositions including a targeting ligand and anexpression-inhibiting oligomeric compound. Disclosed herein arecompositions including a targeting ligand and an RNAi agent.

In some embodiments, the compositions disclosed herein including atargeting ligand and an RNAi agent have the structure represented by:

wherein Z includes or consists of an expression-inhibiting oligomericcompound (Structure 101a);

wherein Z includes or consists of an expression-inhibiting oligomericcompound (Structure 102a); and

wherein Z includes or consists of an expression-inhibiting oligomericcompound (Structure 103a).

Disclosed herein are phosphoramidite compounds including targetingligands.

In some embodiments, the phosphoramidite compounds including targetingligands disclosed herein have the structure represented by:

wherein n is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) (Structure 1d);

Also disclosed are pharmaceutical compositions that include thetargeting ligands disclosed herein.

Disclosed are methods of treating a disease or disorder that wouldbenefit from administration of a therapeutic oligomeric compound, themethod including administering to a subject a therapeutic oligomericcompound linked to a targeting ligand disclosed herein.

Disclosed herein are methods of inhibiting expression of a targetnucleic acid in a subject, the method including administering atherapeutic amount of an expression-inhibiting oligomeric compoundlinked to the targeting ligands disclosed herein.

Disclosed herein are methods of delivering an expression-inhibitingoligomeric compound to the liver in vivo, comprising administering anexpression-inhibiting oligomeric compound linked to a targeting liganddisclosed herein to a subject.

As used herein, the term “linked” when referring to the connectionbetween two molecules means that two molecules are joined by a covalentbond or that two molecules are associated via noncovalent bonds (e.g.,hydrogen bonds or ionic bonds). In some examples, where the term“linked” refers to the association between two molecules via noncovalentbonds, the association between the two different molecules has a K_(D)of less than 1×10⁻⁴ M (e.g., less than 1×10⁻⁵ M, less than 1×10⁻⁶ M, orless than 1×10⁻⁷ M) in physiologically acceptable buffer (e.g.,phosphate buffered saline).

As used herein, the term “directly linked” refers to a first compound orgroup being linked to a second compound or group without any interveningatoms or groups of atoms. As used herein, the term “indirectly linked”refers to a first compound being linked to a second compound or groupthrough an intermediary group, compound, or molecule, such as, forexample, a linking group. Unless otherwise stated, the term “linked” asused herein includes both “directly linked” and “indirectly linked” asthose terms are defined herein.

As used herein, an “oligomeric compound” is a nucleotide sequencecontaining about 10-50 nucleotides or nucleotide base pairs. In someembodiments, an oligomeric compound has a nucleobase sequence that is atleast partially complementary to a coding sequence in an expressedtarget nucleic acid or target gene within a cell. In some embodiments,the oligomeric compounds, upon delivery to a cell expressing a gene, areable to inhibit the expression of the underlying gene, and are referredto herein as “expression-inhibiting oligomeric compounds.” The geneexpression can be inhibited in vitro or in vivo. “Oligomeric compounds”include, but are not limited to: oligonucleotides, single-strandedoligonucleotides, single-stranded antisense oligonucleotides, shortinterfering RNAs (siRNAs), double-strand RNAs (dsRNA), micro RNAs(miRNAs), short hairpin RNAs (shRNA), ribozymes, interfering RNAmolecules, and dicer substrates.

As used herein, the term “oligonucleotide” means a polymer of linkednucleosides each of which can be independently modified or unmodified.

As used herein, the term “single-stranded oligonucleotide” means asingle-stranded oligomeric compound having a sequence at least partiallycomplementary to a target mRNA, that is capable of hybridizing to atarget mRNA through hydrogen bonding under mammalian physiologicalconditions (or comparable conditions in vitro). In some embodiments, asingle-stranded oligonucleotide is a single stranded antisenseoligonucleotide.

As used herein, an “RNAi agent” means an agent that contains an RNA orRNA-like (e.g., chemically modified RNA) oligonucleotide molecule thatis capable of degrading or inhibiting translation of messenger RNA(mRNA) transcripts of a target mRNA in a sequence specific manner. Asused herein, RNAi agents may operate through the RNA interferencemechanism (i.e., inducing RNA interference through interaction with theRNA interference pathway machinery (RNA-induced silencing complex orRISC) of mammalian cells), or by any alternative mechanism(s) orpathway(s). While it is believed that RNAi agents, as that term is usedherein, operate primarily through the RNA interference mechanism, thedisclosed RNAi agents are not bound by or limited to any particularpathway or mechanism of action. RNAi agents include, but are not limitedto: single-stranded oligonucleotides, single-stranded antisenseoligonucleotides, short interfering RNAs (siRNAs), double-strand RNAs(dsRNA), micro RNAs (miRNAs), short hairpin RNAs (shRNA), and dicersubstrates. The RNAi agents described herein are comprised of anoligonucleotide having a strand that is at least partially complementaryto the mRNA being targeted. In some embodiments, the RNAi agentsdescribed herein are double-stranded, and are comprised of an antisensestrand and a sense strand that is at least partially complementary tothe antisense strand. RNAi agents may be comprised of modifiednucleotides and/or one or more non-phosphodiester linkages. In someembodiments, the RNAi agents described herein are single-stranded.

As used herein, the terms “silence,” “reduce,” “inhibit,”“down-regulate,” or “knockdown” when referring to expression of a givengene, mean that the expression of the gene, as measured by the level ofRNA transcribed from the gene or the level of polypeptide, protein orprotein subunit translated from the mRNA in a cell, group of cells,tissue, organ, or subject in which the gene is transcribed, is reducedwhen the cell, group of cells, tissue, organ, or subject is treated witholigomeric compounds linked to the targeting ligands described herein ascompared to a second cell, group of cells, tissue, organ, or subjectthat has not or have not been so treated.

As used herein, the term “sequence” or “nucleotide sequence” mean asuccession or order of nucleobases or nucleotides, described with asuccession of letters using the standard nucleotide nomenclature.

As used herein, and unless otherwise indicated, the term“complementary,” when used to describe a first nucleotide sequence(e.g., RNAi agent sense strand or targeted mRNA) in relation to a secondnucleotide sequence (e.g, single-stranded antisense oligonucleotide or adouble-stranded RNAi agent antisense strand), means the ability of anoligonucleotide or polynucleotide including the first nucleotidesequence to hybridize (form base pair hydrogen bonds under mammalianphysiological conditions (or comparable conditions in vitro)) and form aduplex or double helical structure under certain conditions with anoligonucleotide or polynucleotide including the second nucleotidesequence. Complementary sequences include Watson-Crick base pairs ornon-Watson-Crick base pairs and include natural or modified nucleotidesor nucleotide mimics, at least to the extent that the above requirementswith respect to the ability to hybridize are fulfilled.

As used herein, “perfectly complementary” or “fully complementary” meansthat all (100%) of the bases in a contiguous sequence of a firstpolynucleotide will hybridize with the same number of bases in acontiguous sequence of a second polynucleotide. The contiguous sequencemay comprise all or a part of a first or second nucleotide sequence.

As used herein, “partially complementary” means that in a hybridizedpair of nucleobase sequences, at least 70%, but not all, of the bases ina contiguous sequence of a first polynucleotide will hybridize with thesame number of bases in a contiguous sequence of a secondpolynucleotide.

As used herein, “substantially complementary” means that in a hybridizedpair of nucleobase sequences, at least 85%, but not all, of the bases ina contiguous sequence of a first polynucleotide will hybridize with thesame number of bases in a contiguous sequence of a secondpolynucleotide. The terms “complementary,” “fully complementary,” and“substantially complementary” herein may be used with respect to thebase matching between the sense strand and the antisense strand of adouble-stranded RNAi agent, between the antisense strand of adouble-stranded RNAi agent and a sequence of a target mRNA, or between asingle-stranded antisense oligonucleotide and a sequence of a targetmRNA.

As used herein, the terms “treat,” “treatment,” and the like, mean themethods or steps taken to provide relief from or alleviation of thenumber, severity, and/or frequency of one or more symptoms of a diseasein a subject.

As used herein, the phrase “introducing into a cell,” when referring toan oligomeric compound, means functionally delivering the oligomericcompound into a cell. The phrase “functional delivery,” means thatdelivering the oligomeric compound to the cell in a manner that enablesthe oligomeric compound to have the expected biological activity, e.g.,sequence-specific inhibition of gene expression.

Unless stated otherwise, use of the symbol

as used herein means that any group or groups may be linked thereto thatis in accordance with the scope of the inventions described herein.

As used herein, the term “isomers” refers to compounds that haveidentical molecular formulae, but that differ in the nature or thesequence of bonding of their atoms or in the arrangement of their atomsin space. Isomers that differ in the arrangement of their atoms in spaceare termed “stereoisomers.” Stereoisomers that are not mirror images ofone another are termed “diastereoisomers,” and stereoisomers that arenon-superimposable mirror images are termed “enantiomers,” or sometimesoptical isomers. A carbon atom bonded to four non-identical substituentsis termed a “chiral center.”

As used herein, unless specifically identified in a structure as havinga particular conformation, for each structure in which asymmetriccenters are present and thus give rise to enantiomers, diastereomers, orother stereoisomeric configurations, each structure disclosed herein isintended to represent all such possible isomers, including theiroptically pure and racemic forms. For example, the structures disclosedherein are intended to cover mixtures of diastereomers as well as singlestereoisomers.

The term “substituted,” as used herein, means that any one or morehydrogens on the designated atom, usually a carbon, oxygen, or nitrogenatom, is replaced with any group as defined herein, provided that thedesignated atom's normal valency is not exceeded, and that thesubstitution results in a stable compound. Non-limiting examples ofsubstituents include C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, cyano,hydroxyl, oxo, carboxyl, cycloalkyl, cycloalkenyl, heterocyclyl,heteroaryl, aryl, keto, alkoxycarbonyl, aryloxycarbonyl,heteroaryloxycarbonyl, or halo (e.g., F, Cl, Br, I). When a substituentis keto or oxo (i.e., ═O), then two (2) hydrogens on the atom arereplaced. Ring double bonds, as used herein, are double bonds that areformed between two adjacent ring atoms (e.g., C═C, C═N, N═N, etc.).

Some compounds of the present disclosure can exist in a tautomeric formthat is also intended to be encompassed within the scope of the presentdisclosure. “Tautomers” are compounds whose structures differ markedlyin the arrangement of atoms, but which exist in easy and rapidequilibrium. It is to be understood that compounds of the presentdisclosure may be depicted as different tautomers. It should also beunderstood that when compounds have tautomeric forms, all tautomericforms are intended to be within the scope of the disclosure, and thenaming of the compounds does not exclude any tautomeric form.

The compounds and pharmaceutically acceptable salts of the presentdisclosure can exist in one or more tautomeric forms, includingketone-enol, amide-nitrile, lactam-lactim, amide-imidic acid tautomerismin heterocyclic rings (e.g., in the nucleobases guanine, thymine, andcytosine), amine-enamine and enamine-enamine and geometric isomers andmixtures thereof. Ring-chain tautomerism, exhibited by glucose and othersugars, arises as a result of the aldehyde group (—CHO) in a sugar chainmolecule reacting with one of the hydroxy groups (—OH) in the samemolecule to give it a cyclic (ring-shaped) form. All such tautomericforms are included within the scope of the present disclosure. Tautomersexist as mixtures of a tautomeric set in solution. In solid form,usually one tautomer predominates. Even though one tautomer may bedescribed, the present disclosure includes all tautomers of thecompounds disclosed herein. The concept of tautomers that areinterconvertible by tautomerizations is called tautomerism. Intautomerism, a simultaneous shift of electrons and a hydrogen atomoccurs.

Tautomerizations are catalyzed by: Base: 1. deprotonation; 2. formationof a delocalized anion (e.g. an enolate); 3. protonation at a differentposition of the anion; Acid: 1. protonation; 2. formation of adelocalized cation; 3. deprotonation at a different position adjacent tothe cation.

As used herein, the term “alkyl” refers to a saturated aliphatichydrocarbon group, straight chain or branched, having from 1 to 10carbon atoms unless otherwise specified. For example, “C1-C6 alkyl”includes alkyl groups having 1, 2, 3, 4, 5, or 6 carbons in a linear orbranched arrangement. As used herein, the term “aminoalkyl” refers to analkyl group as defined above, substituted at any position with one ormore amino groups as permitted by normal valency. The amino groups maybe unsubstituted, monosubstituted, or di-substituted.

As used herein, the term “cycloalkyl” means a saturated or unsaturatednonaromatic hydrocarbon ring group having from 3 to 14 carbon atoms,unless otherwise specified. Examples of cycloalkyl groups include, butare not limited to, cyclopropyl, methyl-cyclopropyl,2,2-dimethyl-cyclobutyl, 2-ethyl-cyclopentyl, cyclohexyl, etc.Cycloalkyls may include multiple spiro- or fused rings. Cycloalkylgroups are optionally mono-, di-, tri-, tetra- or penta-substituted onany position as permitted by normal valency.

As used herein, the term “alkenyl” refers to a non-aromatic hydrocarbonradical, straight or branched, containing at least one carbon-carbondouble bond, and having from 2 to 10 carbon atoms unless otherwisespecified. Up to five carbon-carbon double bonds may be present in suchgroups. For example, “C2-C6” alkenyl is defined as an alkenyl radicalhaving from 2 to 6 carbon atoms. Examples of alkenyl groups include, butare not limited to, ethenyl, propenyl, butenyl, and cyclohexenyl. Thestraight, branched, or cyclic portion of the alkenyl group may containdouble bonds and is optionally mono-, di-, tri-, tetra-, orpenta-substituted on any position as permitted by normal valency. Theterm “cycloalkenyl” means a monocyclic hydrocarbon group having thespecified number of carbon atoms and at least one carbon-carbon doublebond.

As used herein, the term “alkynyl” refers to a hydrocarbon radical,straight or branched, containing from 2 to 10 carbon atoms, unlessotherwise specified, and containing at least one carbon-carbon triplebond. Up to 5 carbon-carbon triple bonds may be present. Thus, “C2-C6alkynyl” means an alkynyl radical having from 2 to 6 carbon atoms.Examples of alkynyl groups include, but are not limited to, ethynyl,2-propynyl, and 2-butynyl. The straight or branched portion of thealkynyl group may contain triple bonds as permitted by normal valency,and may be optionally mono-, di-, tri-, tetra-, or penta-substituted onany position as permitted by normal valency.

As used herein, “alkoxyl” or “alkoxy” refers to an alkyl group asdefined above with the indicated number of carbon atoms attached throughan oxygen bridge. C₁₋₆ alkoxy, is intended to include C₁, C₂, C₃, C₄,C₅, and C₆ alkoxy groups. C₁₋₈ alkoxy, is intended to include C₁, C₂,C₃, C₄, C₅, C₆, C₇, and C₈ alkoxy groups. Examples of alkoxy include,but are not limited to, methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy,s-butoxy, t-butoxy, n-pentoxy, s-pentoxy, n-heptoxy, and n-octoxy.

As used herein, “keto” refers to any alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, heterocyclyl, heteroaryl, or aryl group asdefined herein attached through a carbonyl bridge. Examples of ketogroups include, but are not limited to, alkanoyl (e.g., acetyl,propionyl, butanoyl, pentanoyl, hexanoyl), alkenoyl (e.g., acryloyl)alkynoyl (e.g., ethynoyl, propynoyl, butynoyl, pentynoyl, hexynoyl),aryloyl (e.g., benzoyl), heteroaryloyl (e.g., pyrroloyl, imidazoloyl,quinolinoyl, pyridinoyl).

As used herein, “alkoxycarbonyl” refers to any alkoxy group as definedabove attached through a carbonyl bridge (i.e., —C(O)O-alkyl). Examplesof alkoxycarbonyl groups include, but are not limited to,methoxycarbonyl, ethoxycarbonyl, iso-propoxycarbonyl, n-propoxycarbonyl,t-butoxycarbonyl, benzyloxycarbonyl or n-pentoxycarbonyl.

As used herein, “aryloxycarbonyl” refers to any aryl group as definedherein attached through an oxycarbonyl bridge (i.e., —C(O)O-aryl).Examples of aryloxycarbonyl groups include, but are not limited to,phenoxycarbonyl and naphthyloxycarbonyl.

As used herein, “heteroaryloxycarbonyl” refers to any heteroaryl groupas defined herein attached through an oxycarbonyl bridge (i.e.,—C(O)O-heteroaryl). Examples of heteroaryloxycarbonyl groups include,but are not limited to, 2-pyridyloxycarbonyl, 2-oxazolyloxycarbonyl,4-thiazolyloxycarbonyl, or pyrimidinyloxycarbonyl.

As used herein, “aryl” or “aromatic” means any stable monocyclic orpolycyclic carbon ring of up to 7 atoms in each ring, wherein at leastone ring is aromatic. Examples of aryl groups include, but are notlimited to, phenyl, naphthyl, anthracenyl, tetrahydronaphthyl, indanyl,and biphenyl. In cases where the aryl substituent is bicyclic and onering is non-aromatic, it is understood that attachment is via thearomatic ring. Aryl groups are optionally mono-, di-, tri-, tetra-, orpenta-substituted on any position as permitted by normal valency.

As used herein, the term “heteroaryl” represents a stable monocyclic orpolycyclic ring of up to 7 atoms in each ring, wherein at least one ringis aromatic and contains from 1 to 4 heteroatoms selected from the groupconsisting of O, N and S. Examples of heteroaryl groups include, but arenot limited to, acridinyl, carbazolyl, cinnolinyl, quinoxalinyl,pyrrazolyl, indolyl, benzotriazolyl, furanyl, thienyl, benzothienyl,benzofuranyl, benzimidazolonyl, benzoxazolonyl, quinolinyl,isoquinolinyl, dihydroisoindolonyl, imidazopyridinyl, isoindolonyl,indazolyl, oxazolyl, oxadiazolyl, isoxazolyl, indolyl, pyrazinyl,pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl, tetrahydroquinoline.“Heteroaryl” is also understood to include the N-oxide derivative of anynitrogen-containing heteroaryl. In cases where the heteroarylsubstituent is bicyclic and one ring is non-aromatic or contains noheteroatoms, it is understood that attachment is via the aromatic ringor via the heteroatom containing ring. Heteroaryl groups are optionallymono-, di-, tri-, tetra-, or penta-substituted on any position aspermitted by normal valency.

As used herein, the term “heterocycle,” “heterocyclic,” or“heterocyclyl” means a 3- to 14-membered aromatic or nonaromaticheterocycle containing from 1 to 4 heteroatoms selected from the groupconsisting of O, N and S, including polycyclic groups. As used herein,the term “heterocyclic” is also considered to be synonymous with theterms “heterocycle” and “heterocyclyl” and is understood as also havingthe same definitions set forth herein. “Heterocyclyl” includes the abovementioned heteroaryls, as well as dihydro and tetrahydro analogsthereof. Examples of heterocyclyl groups include, but are not limitedto, azetidinyl, benzoimidazolyl, benzofuranyl, benzofurazanyl,benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl,carbazolyl, carbolinyl, cinnolinyl, furanyl, imidazolyl, indolinyl,indolyl, indolazinyl, indazolyl, isobenzofuranyl, isoindolyl,isoquinolyl, isothiazolyl, isoxazolyl, naphthpyridinyl, oxadiazolyl,oxooxazolidinyl, oxazolyl, oxazoline, oxopiperazinyl, oxopyrrolidinyl,oxomorpholinyl, isoxazoline, oxetanyl, pyranyl, pyrazinyl, pyrazolyl,pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl, pyridinonyl,pyrimidyl, pyrimidinonyl, pyrrolyl, quinazolinyl, quinolyl,quinoxalinyl, tetrahydropyranyl, tetrahydrofuranyl,tetrahydrothiopyranyl, tetrahydroisoquinolinyl, tetrazolyl,tetrazolopyridyl, thiadiazolyl, thiazolyl, thienyl, triazolyl,1,4-dioxanyl, hexahydroazepinyl, piperazinyl, piperidinyl,pyridin-2-onyl, pyrrolidinyl, morpholinyl, thiomorpholinyl,dihydrobenzoimidazolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl,dihydrobenzoxazolyl, dihydrofuranyl, dihydroimidazolyl, dihydroindolyl,dihydroisooxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl,dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl,dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl,dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl,dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl,dioxidothiomorpholinyl, methylenedioxybenzoyl, tetrahydrofuranyl, andtetrahydrothienyl, and N-oxides thereof. Attachment of a heterocyclylsubstituent can occur via a carbon atom or via a heteroatom.Heterocyclyl groups are optionally mono-, di-, tri-, tetra-, orpenta-substituted on any position as permitted by normal valency.

The person of ordinary skill in the art would readily understand andappreciate that the compounds and compositions disclosed herein may havecertain atoms (e.g., N, O, or S atoms) in a protonated or deprotonatedstate, depending upon the environment in which the compound orcomposition is placed. Accordingly, as used herein, the structuresdisclosed herein envisage that certain functional groups, such as, forexample, OH, SH, or NH, may be protonated or deprotonated. Thedisclosure herein is intended to cover the disclosed compounds andcompositions regardless of their state of protonation based on the pH ofthe environment, as would be readily understood by the person ofordinary skill in the art.

As used in a claim herein, the phrase “consisting of” excludes anyelement, step, or ingredient not specified in the claim. When used in aclaim herein, the phrase “consisting essentially of” limits the scope ofa claim to the specified materials or steps and those that do notmaterially affect the basic and novel characteristic(s) of the claimedinvention.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a ¹H NMR spectra of compound 3 (which is described below inExample 1).

FIG. 2 is a ¹H NMR spectra of compound 4 (which is described below inExample 1).

FIG. 3 is a ¹H NMR spectra of compound 6 (which is described below inExample 1)

FIG. 4 is a ¹H NMR spectra of compound 7 (which is described below inExample 1)

FIG. 5 is a ¹H NMR spectra of compound 9 (which is described below inExample 1).

FIG. 6 is a ¹H NMR spectra of compound 10 (which is Structure 101dherein, and is described below in Example 1).

FIG. 7 is a ¹H NMR spectra of compound 13 (which is Structure 103dherein, and is described below in Example 2).

FIG. 8 is a ¹H NMR spectra of compound 16 (which is Structure 102dherein, and is described below in Example 3).

FIG. 9 is a HPLC chromatograph for AM03704 conjugated to Structure 103d(which is described below in Example 5).

FIG. 10 is a HPLC chromatograph for AM03704 conjugated to Structure 101d(which is described below in Example 5).

FIG. 11 is a HPLC chromatograph for AM03704 conjugated to Structure 102d(which is described below in Example 5).

FIG. 12 is a graph illustrating normalized mouse Factor 12 (mF12)protein levels in wild type mice (which is described below in Example6).

FIG. 13 is a graph illustrating normalized mouse Factor 12 (F12) proteinlevels in wild type mice (which is described below in Example 7).

FIG. 14 is a graph illustrating normalized lipoprotein(a) (Lp(a))particle levels in Lp(a) transgenic (Tg) mice (which is described belowin Example 8).

FIG. 15 is a graph illustrating normalized lipoprotein(a) (Lp(a))particle levels in Lp(a) Tg mice (which is described below in Example9).

FIG. 16 is a graph illustrating normalized apo(a) levels in apo(a)transgenic (Tg) mice (which is described below in Example 10).

FIG. 17 is a graph illustrating normalized cF12 protein levels incynomolgus monkeys (which is described below in Example 12).

FIG. 18 is a graph illustrating normalized AAT (Z-AAT) protein levels inPiZ transgenic mice (which is described below in Example 13).

FIG. 19 is a graph illustrating normalized mouse Factor 12 (F12) proteinlevels in wild type mice (which is described below in Example 14).

FIG. 20 is a formula (Formula A) that represents the general structureof targeting ligands disclosed herein.

DETAILED DESCRIPTION

Described herein are novel targeting ligands that are linked tocompounds, such as therapeutic or diagnostic expression-inhibitingoligomeric compounds. In some embodiments, the compounds that are linkedto the targeting ligands described herein include or consist oftherapeutic compounds that are RNAi agents. The targeting ligands can beused to target therapeutic compounds to a desired location of a targetnucleic acid or target gene. Also described herein are compositionsincluding targeting ligands and therapeutic compounds, such ascompositions including or consisting of targeting ligands andexpression-inhibiting oligomeric compounds.

The new targeting ligands disclosed herein provide efficient targetingor bio-distribution, sufficient stability in vivo and in vitro, and aresuitable for synthesis as phosphoramidites, which reduces the cost andburden of manufacture, and can increase efficacy over previouslyconsidered targeting ligands linked to an expression-inhibitingoligomeric compound, such as an RNAi agent.

Targeting Ligands

Targeting ligands are comprised of one or more targeting group(s) ortargeting moiety(ies), which can serve to enhance the pharmacokinetic orbio-distribution properties of the compound to which they are linked,and improve cell- or tissue-specific distribution and cell-specificuptake of the conjugated composition. In general, a targeting ligandaids in directing the delivery of the therapeutic compound to which itis linked to the desired target site. In some instances, the targetingmoiety may bind to a cell or cell receptor, and initiate endocytosis tofacilitate entry of the therapeutic compound into the cell. Targetingmoieties can include compounds with affinity to cell receptors or cellsurface molecules or antibodies. A variety of targeting ligands thatcontain targeting moieties can be linked to therapeutic agents and othercompounds to target the agents to cells and specific cellular receptors.Types of targeting moieties include carbohydrates, cholesterol andcholesteryl groups, and steroids. Targeting moieties that can bind tocell receptors include saccharides, such as galactose, galactosederivatives (such as N-acetyl-galactosamine), mannose, and mannosederivatives; other carbohydrates; glycans; haptens; vitamins; folate;biotin; aptamers; and peptides, such as RGD-containing peptides,insulin, EGF, and transferrin.

Targeting moieties that are known to bind to the asialoglycoproteinreceptor (ASGPR) are particularly useful in directing the delivery ofoligomeric compounds to the liver. Asialoglycoprotein receptors areabundantly expressed on liver cells, including hepatocytes. Cellreceptor targeting moieties that target ASGPR include galactose andgalactose derivatives. In particular, clusters of galactose derivatives,including clusters comprised of two, three, or fourN-acetyl-galactosamines (GalNAc or NAG), can facilitate uptake ofcertain compounds in liver cells. GalNAc clusters conjugated tooligomeric compounds serve to direct the composition to the liver, wherethe N-acetyl-galactosamine sugars are able to bind to theasialoglycoprotein receptors on the surface of the liver cell. Thebinding to an asialoglycoprotein receptor is believed to initiatereceptor-mediated endocytosis, thereby facilitating entry of thecompound into the interior of the cell.

The targeting ligands disclosed herein may include one, two, three,four, or more than four targeting moieties. In some embodiments, thetargeting ligands disclosed herein can include one, two, three, four, ormore than four targeting moieties linked to a branch point group. Insome embodiments, the targeting ligands disclosed herein can includeone, two, three, four, or more than four targeting moieties linked to abranch point group wherein each targeting moiety is linked to the branchpoint group via a tether.

In some embodiments, the targeting ligands disclosed herein can includeone, two, three, four, or more than four asialoglycoprotein receptor(ASGPR) targeting moieties linked to a branch point group. In someembodiments, the targeting ligands disclosed herein can include one,two, three, four, or more than four ASGPR targeting moieties linked to abranch point group wherein each ASGPR targeting moiety is linked to thebranch point group via a tether.

The targeting ligands described herein are represented by the followingFormula A of FIG. 20, wherein n is an integer from 1 to 4 (e.g., 1, 2,3, or 4) (Formula A). In some embodiments, n in Formula A is an integerfrom 1-3, 1-2, 2-4, 2-3, or 3-4.

The targeting ligands disclosed herein can be linked to therapeuticcompounds, such as oligomeric compounds. In some embodiments, thetargeting ligand is linked to the therapeutic compound via an additionallinker and/or a cleavable moiety, which is then linked to thetherapeutic compound. In some embodiments, targeting ligands are ligatedto the therapeutic compound itself.

In some embodiments, the therapeutic compound is anexpression-inhibiting oligomeric compound. In some embodiments, theexpression-inhibiting oligomeric compound is an RNAi agent. In someembodiments, the expression-inhibiting oligomeric compound is adouble-stranded RNAi agent.

In some embodiments, a targeting ligand is linked directly or indirectlyto the 5′ end of the sense strand of a double-stranded RNAi agent. Insome embodiments, the targeting ligand is linked directly or indirectlyto the 3′ end of the sense strand of a double-stranded RNAi agent. Insome embodiments, the targeting ligand is linked directly or indirectlyto the 5′ end or the 3′ end of the antisense strand of a double-strandedRNAi agent. In some embodiments, the targeting ligand is linked directlyor indirectly to the 5′ end or the 3′ end of a single-stranded RNAiagent.

In some embodiments, a targeting ligand is linked to a double-strandedRNAi agent via a phosphate, phosphonate, phosphorothioate, or otherinternucleoside linking group, at the 5′ end of the terminal nucleosideof the sense strand of the double-stranded RNAi agent.

In some embodiments, a targeting ligand disclosed herein includes acleavable moiety. In some embodiments, a cleavable moiety includes orconsists of a phosphate or other internucleoside linking group that maybe cleaved. In some embodiments, the targeting ligand is linked to atherapeutic compound via a cleavable moiety.

In some embodiments, a targeting ligand disclosed herein is linked to anadditional group or groups that includes a cleavable moiety. In someembodiments, the targeting ligand is linked to a cleavable moiety, whichis then linked to an expression-inhibiting oligomeric compound.

In some embodiments, the targeting ligand is a phosphoramidite compound(also referred to herein as a “phosphoramidite-containing compound”). Aphosphoramidite compound including a targeting ligand described hereinmay be useful to readily attach the targeting ligand to the therapeuticcompound or to other groups, using methods generally known in the artfor phosphoramidite synthesis. In some embodiments, the phosphoramiditecompound including the targeting ligand is linked to anexpression-inhibiting oligomeric compound using methods generally knownin the art. In some embodiments, the targeting ligand-containingphosphoramidite is linked to the 5′ end of the sense strand of adouble-stranded RNAi agent.

In some embodiments, an expression-inhibiting oligomeric compound linkedto a targeting ligand includes a single-stranded oligonucleotide. Insome embodiments, the single-stranded oligonucleotide is asingle-stranded antisense oligonucleotide. In some embodiments, thetargeting ligand is linked directly to a single-stranded antisenseoligonucleotide. In some embodiments, additional groups are insertedbetween a targeting ligand and a single-stranded oligonucleotide.

In some embodiments, the targeting ligand linked to an RNAi agentincludes one or more N-acetyl-galactosamine sugars as a targeting moietyor targeting moieties.

In some embodiments, the targeting ligand linked to anexpression-inhibiting oligomeric compound includes a tether thatincludes polyethylene glycol (PEG). In some embodiments, a tetherconsists of PEG. In some embodiments a tether includes a PEG having 1 to10 ethylene glycol units. In some embodiments a tether includes a PEGhaving 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ethylene glycol units.

In some embodiments, the targeting ligand linked to the RNAi agentcomprises polyethylene glycol (PEG) as the linker. In some embodiments,the linker comprises PEG. In some embodiments, the linker consists ofPEG. In some embodiments a linker comprises a PEG having 1 to 20ethylene glycol units. In some embodiments a tether comprises a PEGhaving 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20 ethylene glycol units.

In some embodiments, an expression-inhibiting oligomeric compound linkedto any of the targeting ligands disclosed herein includes an RNAi agent.In some embodiments, a targeting ligand disclosed herein is linked,either directly or indirectly, to an RNAi agent.

In some embodiments, a targeting ligand disclosed herein is linkeddirectly to an RNAi agent. In some embodiments, a targeting liganddisclosed herein is linked indirectly to an RNAi agent, as additionalgroup(s) are inserted between the RNAi agent and the linker of thetargeting ligand. In some embodiments, a second linker is includedbetween the linker and the therapeutic compound.

Targeting Ligand Structures, and Phosphoramidite Compounds IncludingTargeting Ligands.

The targeting ligands disclosed herein may be comprised of one or moretargeting moieties, tethers, branch point groups, and linkers. Thetargeting ligands disclosed herein may contain one, two, three, four, ormore than four targeting moieties.

In some embodiments, the targeting ligands disclosed herein aresynthesized to be in the form of a phosphoramidite compound.Phosphoramidites are widely used in the chemical synthesis of RNA andDNA. In some embodiments, the phosphoramidite-containing targetingligands disclosed herein are added to the 5′ end of the sense strand ofa double-stranded RNAi agent. It can be especially advantageous toprepare the targeting ligand as a phosphoramidite when the targetingligand is to be linked to the 5′ terminal end of anexpression-inhibiting oligomeric compound. Not wishing to be bound bytheory, it is understood that preparing the targeting ligand as aphosphoramidite when the targeting ligand is linked to the 5′ terminalend of an expression-inhibiting oligomeric compound not only allows forthe linkage of the targeting ligand as the last component (thus reducingmanufacturing costs), as well as potentially permits the targetingligand to block the loading of the sense strand into RISC when thetargeting ligand is attached to the 5′ terminal end of the sense strandof a double-stranded RNAi agent. When an expression-inhibitingoligomeric compound is a double-stranded RNAi agent, the targetingligand can be prepared as a phosphoramidite compound when the targetingligand is to be linked to the 5′ terminal end of the sense strand of theRNAi agent.

In some embodiments, the targeting ligand is represented by thefollowing Formula B:

wherein n is an integer from 1 to 20; X is O, S, or NH; and TargetingMoiety is selected from the group consisting of galactose,galactosamine, N-formyl-galactosamine, N-acetyl-galactosamine,N-propionyl-galactosamine, N-n-butanoylgalactosamine, orN-iso-butanoylgalactosamine. (Formula B). In some embodiments, n equals6. In some embodiments, n equals 8. In some embodiments, n equals 4.

In some embodiments, the targeting ligand has the structure representedby the following:

wherein n is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) (Structure 1).

In some embodiments, the targeting ligand has the structure representedby Structure 1, wherein n=6. In some embodiments, the targeting ligandhas the structure represented by Structure 1, wherein n=8. In someembodiments, the targeting ligand has the structure represented byStructure 1, wherein n=4.

In some embodiments, the targeting ligand is linked to anexpression-inhibiting oligomeric compound, and has the structurerepresented by the following:

wherein Z includes or consists of an expression-inhibiting oligomericcompound (Structure 1a).

In some embodiments, the targeting ligand is linked to anexpression-inhibiting oligomeric compound, and has the structurerepresented by the following:

wherein Z consists of or includes an expression-inhibiting oligomericcompound (Structure 1b).

In some embodiments, the targeting ligand is linked to anexpression-inhibiting oligomeric compound, and has the structurerepresented by the following:

wherein Z consists of or includes an expression-inhibiting oligomericcompound (Structure 1c).

In some embodiments, the targeting ligand is aphosphoramidite-containing compound having the structure represented bythe following:

wherein n is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) (Structure 1d).

In some embodiments, the targeting ligand comprises or consists of thestructure represented by the following:

In some embodiments, the targeting ligand is linked to anexpression-inhibiting oligomeric compound, and has the structurerepresented by the following:

wherein Z includes or consists of an expression-inhibiting oligomericcompound (Structure 101a).

In some embodiments, the targeting ligand is linked to anexpression-inhibiting oligomeric compound, and has the structurerepresented by the following:

wherein Z consists of or includes an expression-inhibiting oligomericcompound (Structure 101b).

In some embodiments, the targeting ligand is linked to anexpression-inhibiting oligomeric compound, and has the structurerepresented by the following:

wherein Z consists of or includes an expression-inhibiting oligomericcompound (Structure 101c).

In some embodiments, the targeting ligand is aphosphoramidite-containing compound having the structure represented bythe following:

In some embodiments, the targeting ligand comprises or consists of thestructure represented by the following:

In some embodiments, the targeting ligand is linked to anexpression-inhibiting oligomeric compound, and has the structurerepresented by the following:

wherein Z includes or consists of an expression-inhibiting oligomericcompound (Structure 102a).

In some embodiments, the targeting ligand is linked to anexpression-inhibiting oligomeric compound, and has the structurerepresented by the following:

wherein Z consists of or includes an expression-inhibiting oligomericcompound (Structure 102b).

In some embodiments, the targeting ligand is linked to anexpression-inhibiting oligomeric compound, and has the structurerepresented by the following:

wherein Z consists of or includes an expression-inhibiting oligomericcompound (Structure 102c).

In some embodiments, the targeting ligand is aphosphoramidite-containing compound having the structure represented bythe following:

In some embodiments, the targeting ligand comprises or consists of thestructure represented by the following:

In some embodiments, the targeting ligand is linked to anexpression-inhibiting oligomeric compound, and has the structurerepresented by the following:

wherein Z includes or consists of an expression-inhibiting oligomericcompound (Structure 103a).

In some embodiments, the targeting ligand is linked to anexpression-inhibiting oligomeric compound, and has the structurerepresented by the following:

wherein Z consists of or includes an expression-inhibiting oligomericcompound (Structure 103b).

In some embodiments, the targeting ligand is linked to anexpression-inhibiting oligomeric compound, and has the structurerepresented by the following:

wherein Z consists of or includes an expression-inhibiting oligomericcompound (Structure 103c).

In some embodiments, the targeting ligand is aphosphoramidite-containing compound having the structure represented bythe following:

In some embodiments, the targeting ligand has the structure representedby the following:

In some embodiments, an expression-inhibiting oligomeric compound islinked to the targeting ligand and has the structure represented by thefollowing:

wherein Z consists of or includes an expression-inhibiting oligomericcompound; A is O or S; and A′ is O⁻, S⁻, or NH⁻ (Structure 2b).

In some embodiments, the targeting ligand is aphosphoramidite-containing compound having the structure represented bythe following:

In some embodiments, the targeting ligand has the structure representedby the following:

In some embodiments, an expression-inhibiting oligomeric compound islinked to the targeting ligand and has the structure represented by thefollowing:

wherein Z consists of or includes an expression-inhibiting oligomericcompound; A is O or S; and A′ is O⁻, S⁻, or NH⁻ (Structure 3b).

In some embodiments, the targeting ligand is aphosphoramidite-containing compound having the structure represented bythe following:

In some embodiments, the targeting ligand has the structure representedby the following:

In some embodiments, an expression-inhibiting oligomeric compound islinked to the targeting ligand and has the structure represented by thefollowing:

wherein Z consists of or includes an expression-inhibiting oligomericcompound; A is O or S; and A′ is O⁻, S⁻, or NH⁻ (Structure 4b).

In some embodiments, the targeting ligand is aphosphoramidite-containing compound having the structure represented bythe following:

In some embodiments, the targeting ligand has the structure representedby the following:

In some embodiments, an expression-inhibiting oligomeric compound islinked to the targeting ligand and has the structure represented by thefollowing:

wherein Z consists of or includes an expression-inhibiting oligomericcompound; A is O or S; and A′ is O⁻, S⁻, or NH⁻ (Structure 5b).

In some embodiments, the targeting ligand is aphosphoramidite-containing compound having the structure represented bythe following:

In some embodiments, the targeting ligand has the structure representedby the following:

In some embodiments, an expression-inhibiting oligomeric compound islinked to the targeting ligand and has the structure represented by thefollowing:

wherein Z consists of or includes an expression-inhibiting oligomericcompound; A is O or S; and A′ is O⁻, S⁻, or NH⁻ (Structure 6b).

In some embodiments, the targeting ligand is aphosphoramidite-containing compound having the structure represented bythe following:

In some embodiments, the targeting ligand is in the form of a galactosecluster. As used herein, a galactose cluster includes a targeting ligandhaving two to four terminal galactose derivatives. As used herein, theterm galactose derivative includes both galactose and derivatives ofgalactose having affinity for the asialoglycoprotein receptor equal toor greater than that of galactose. A galactose derivative is asaccharide sugar that is a type of targeting moiety. A terminalgalactose derivative may be linked to a tether through the C-1 carbon ofthe saccharide.

In some embodiments, the targeting ligand is comprised of three terminalgalactosamines or galactosamine derivatives (such asN-acetyl-galactosamine) each having affinity for the asialoglycoproteinreceptor. In some embodiments, the targeting ligand includes threeterminal N-acetyl-galactosamines (GalNAc or NAG) as the targetingmoieties.

In some embodiments, the targeting ligand is comprised of four terminalgalactosamines or galactosamine derivatives (such asN-acetyl-galactosamine) each having affinity for the asialoglycoproteinreceptor. In some embodiments, the targeting ligand includes fourterminal N-acetyl-galactosamines (GalNAc or NAG) as the targetingmoieties.

In some embodiments, each targeting moiety includes a galactosaminederivative that is N-acetyl-galactosamine. Other saccharides havingaffinity for the asialoglycoprotein receptor that may be used astargeting moieties may be selected from the list including: galactose,galactosamine, N-formyl-galactosamine, N-acetyl-galactosamine,N-propionyl-galactosamine, N-n-butanoylgalactosamine, andN-iso-butanoylgalactosamine. The affinities of numerous galactosederivatives for the asialoglycoprotein receptor have been studied (seefor example: Iobst, S. T. and Drickamer, K. J.B.C. 1996, 271, 6686) orare readily determined using methods well known and commonly used in theart.

Terms commonly used in the art when referring to three terminalN-acetyl-galactosamines include tri-antennary, tri-valent, and trimer.

Linkers

The targeting ligands disclosed herein comprise a linker.

The linker is a group of atoms linked to a branch point group on oneend, and linked to a therapeutic compound (or to the phosphorus atom ofa phosphoramidite through a phosphitylation reaction with aphosphoramidite forming reagent, when the targeting ligand issynthesized as a phosphoramidite compound) on the other end. In someembodiments, the linker is linked to a branch point group on one end,and is ligated on the other end to a group or groups that are thenligated to an expression-inhibiting oligomeric compound. In someembodiments, the linker is directly linked to an oligomeric compound. Insome embodiments, the linker is linked to a cleavable moiety, which isthen linked to an oligomeric compound. Examples of cleavable moietiesinclude, for example, phosphate groups, groups including a disulfidemoiety, and/or other internucleoside linkages that may be cleaved. Insome embodiments, the linker is not linked to a cleavable moiety. Insome embodiments, the linker is linked to a phosphorothioate orphosphonate group.

In some embodiments, the linker consists of or includes a polyethyleneglycol (“PEG”) moiety. Incorporating a PEG moiety into the linkerconfers certain beneficial properties over certain other linkers, suchas linkers that consist of or include substituted or unsubstituted alkylchains. For example, incorporating a PEG moiety into the linkerincreases the solubility of the targeting ligand-containingphosphoramidite compound in solvents commonly used in nucleotidesynthesis as compared to compounds that contain alkyl chain linkers,which can lead to simplified manufacturing processes.

In some embodiments, a targeting ligand comprises a linker having thefollowing structure:

wherein n is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) (Structure 1001).

In some embodiments, a targeting ligand comprises a linker linked to aphosphate group having the following structure:

wherein n is an integer selected from 1 to 20. (Structure 1002).

In some embodiments, a targeting ligand comprises a linker linked to aphosphorothioate group having the following structure:

wherein n is an integer selected from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) (Structure1003).

In some embodiments, a targeting ligand comprises a linker having thefollowing structure:

In some embodiments, a targeting ligand comprises a linker linked to aphosphate group having the following structure:

In some embodiments, a targeting ligand comprises a linker linked to aphosphorothioate group having the following structure:

In some embodiments, the linker is linked to an expression-inhibitingoligomeric compound that is a double-stranded RNAi agent. In someembodiments, the linker is linked to the 5′ end of the sense strand of adouble-stranded RNAi agent. In some embodiments, the linker is linked tothe 3′ end of the sense strand of a double-stranded RNAi agent. In someembodiments the linker is linked to the 3′ end of the antisense strandof a double-stranded RNAi agent. In some embodiments, the linker islinked to the 5′ end of the antisense strand of a double-stranded RNAiagent.

In some embodiments, the linker is linked to a cleavable moiety. In someembodiments, a terminal phosphate group of an expression-inhibitingoligomeric compound can serve as a cleavable moiety. In someembodiments, an independently selected cleavable moiety is linked to alinker. As used herein, a cleavable moiety is a group that is stableoutside of the cell, but upon entry into the target cell is cleaved.Cleavable moieties are susceptible to cleavage under certain conditions,such as pH, or certain cleavage agents, such as molecules that promotedegradation or redox agents.

In some embodiments, the cleavable moiety may be susceptible to pH. Forexample, endosomes and lysosomes are known to generally have a moreacidic pH (pH of approximately 4.5 to 6.5) than human blood (pH ofapproximately 7.35 to 7.45), and as such may promote the cleavage of acleavable moiety.

In some embodiments, a cleavable moiety is a phosphate group. Phosphategroups may be cleaved by agents that are known to degrade or hydrolyzephosphate groups.

Branch Point Groups

The targeting ligands disclosed herein comprise at least one branchpoint group. The branch point group of the targeting ligands disclosedherein is attached to a linker. In some embodiments, the branch pointgroup of the targeting ligands disclosed herein is linked to a linker onone end, and the branch point group is linked to one or more tethers onthe other end(s). In some embodiments, the branch point group isattached to a linker and one or more tethers. In some embodiments, thebranch point group is attached indirectly (e.g., via the linker) to anexpression-inhibiting oligomeric compound. In some embodiments, thebranch point group is linked to an expression-inhibiting oligomericcompound via an additional group or groups.

The branch point groups disclosed herein can be of any group whichpermits attachment of one or more targeting moieties and further permitsattachment to a linker.

The branch point groups disclosed herein can be of any group whichpermits attachment of two, three, or four galactose derivatives andfurther permits attachment of the branch point to a linker.

In some embodiments, the targeting ligand comprises a branch pointhaving the following structures:

Tethers

The targeting ligands disclosed herein comprise one or more tethers. Atether is linked between the branch point group and each targetingmoiety. In some embodiments, the tether is linked directly to thetargeting ligand on one end and directly to the branch point group onthe other end. In some embodiments, the tether is linked directly to thetargeting ligand on one end, and indirectly to the branch point group onthe other end. In some embodiments, the tether is linked indirectly tothe targeting ligand on one end and indirectly to the branch point groupon the other end. In some embodiments, a targeting ligand describedherein includes three tethers and three targeting moieties. In someembodiments, a targeting ligand described herein includes four tethersand four targeting moieties. In some embodiments, a targeting liganddescribed herein includes one tether and one targeting moiety. In someembodiments, a targeting ligand described herein includes multipletethers and multiple targeting moieties.

In some embodiments, additional tethers or other groups are insertedbetween the tether and the targeting moiety. In some embodiments, asecond tether is inserted between a tether and a targeting moiety. Insome embodiments, a second tether and a third tether is inserted betweena tether and a targeting moiety. In some embodiments, a second, third,and fourth tether is inserted between a tether and a targeting moiety.As disclosed herein, there is at least one tether present for everytargeting moiety. In some embodiments, there is more than one tetherpresent for each targeting moiety. The targeting ligands disclosedherein are intended to cover such compositions.

In some embodiments, additional groups can be inserted between thetether and the branch point group.

As disclosed herein, the tether serves as a spacer that may further addflexibility and/or length to the linkage between the targeting moietyand the branch point group, linker, and therapeutic compound. In someembodiments, the tether includes alkyl groups (including cycloalkylgroups), alkenyl groups (including cycloalkenyl groups), alkynyl groups,aryl groups, aralkyl groups, aralkenyl groups, or aralkynyl groups. Insome embodiments, the tether includes one or more heteroatoms,heterocycles, heteroaryls, amino acids, nucleotides, or saccharides.

In some embodiments, the targeting ligand includes a tether having thefollowing structure:

wherein n is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20), and X is O, S, or NH(Structure 301).

In some embodiments, the targeting ligand includes a tether having thefollowing structure:

wherein X is O, S, or NH (Structure 302).

In some embodiments, the targeting ligand includes a tether having thefollowing structure:

In some embodiments, the targeting ligand includes a tether having thefollowing structure:

wherein n is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20), and X is O, S, or NH.(Structure 303).

In some embodiments, the targeting ligand includes a tether having thefollowing structure:

wherein n is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20), and X is O, S, or NH.(Structure 304).

In some embodiments, the targeting ligand includes a tether having thefollowing structure:

wherein X is O, S, or NH (Structure 305).

In some embodiments, the targeting ligand includes a tether having thefollowing structure:

wherein X is O, S, or NH (Structure 306).

In some embodiments, the targeting ligand includes more than one type oftether. In some embodiments, the tether acts as a flexible hydrophilicspacer (See, for example, U.S. Pat. No. 5,885,968; and Biessen et al. J.Med. Chem. 1995, 39, 1538-1546, both of which are incorporated herein byreference in their entirety), and includes a PEG spacer. In otherembodiments, the PEG spacer has 1 to 20 ethylene units (PEG₁ to PEG₂₀).For example, the PEG spacer has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20 ethylene units.

Targeting Moieties:

The targeting ligands disclosed herein can include one to four, or morethan four, targeting moieties.

In some embodiments, the targeting ligands may be a galactose cluster.As used herein, a galactose cluster includes a targeting ligand havingtwo to four terminal galactose derivatives. As used herein, the termgalactose derivative includes both galactose and derivatives ofgalactose having affinity for the asialoglycoprotein receptor equal toor greater than that of galactose. A galactose derivative is asaccharide sugar that is a type of targeting moiety. A terminalgalactose derivative is linked to a tether through the C-1 carbon of thesaccharide.

In some embodiments, the targeting ligand is comprised of three terminalgalactosamines or galactosamine derivatives (such asN-acetyl-galactosamine) each having affinity for the asialoglycoproteinreceptor. In some embodiments, the targeting ligand includes threeterminal N-acetyl-galactosamines (GalNAc or NAG) as the targetingmoieties. For example, each of Structures 1, 101, 102, and 103 aretargeting ligands having three terminal N-acetyl-galactosamines as thetargeting moieties.

In some embodiments, each targeting moiety includes a galactosaminederivative that is N-acetyl-galactosamine. Other saccharides havingaffinity for the asialoglycoprotein receptor that may be used astargeting moieties may be selected from the list including: galactose,galactosamine, N-formyl-galactosamine, N-propionyl-galactosamine,N-n-butanoylgalactosamine, and N-iso-butanoylgalactosamine. Theaffinities of numerous galactose derivatives for the asialoglycoproteinreceptor have been studied (see, for example, Iobst, S. T. andDrickamer, K. J.B.C. 1996, 271, 6686, which is incorporated by referenceherein in its entirety) or are readily determined using methods wellknown and commonly used in the art.

In some embodiments, the targeting moiety is a cell-targeting moiety.

In some embodiments, the targeting moiety includes anN-acetyl-galactosamine:

In some embodiments, the targeting ligand includes three targetingmoieties. In some embodiments, the targeting ligand includes fourtargeting moieties. In some embodiments, the targeting ligand includesone targeting moiety. In some embodiments, the targeting ligand includestwo targeting moieties. In some embodiments, the targeting ligandincludes four or more targeting moieties.

In some embodiments, the targeting moiety includes one or more ofgalactose, galactosamine, N-formyl-galactosamine,N-acetyl-galactosamine, N-propionyl-galactosamine,N-n-butanoylgalactosamine, or N-iso-butanoylgalactosamine.

For example, in some embodiments, the N-acetyl-galactosamine targetingmoieties in any of Structures 1 through 6 can be replaced withalternative targeting moieties. In some embodiments, theN-acetyl-galactosamine targeting moieties in any of Structures 101, 102or 103 can be replaced with alternative targeting moieties. Suchalternative targeting moieties include, for example, galactose,galactosamine, N-formyl-galactosamine, N-acetyl-galactosamine,N-propionyl-galactosamine, N-n-butanoylgalactosamine, orN-iso-butanoylgalactosamine.

Additionally, in some embodiments, the targeting moieties of Structures1 through 6 may be replaced with, for example, other carbohydrates;glycans; haptens; vitamins; folate; biotin; aptamers; and/or peptides,such as RGD-containing peptides, insulin, EGF, and/or transferrin. Insome embodiments, the targeting moieties of Structures 101, 102, or 103may be replaced with, for example, other carbohydrates; glycans;haptens; vitamins; folate; biotin; aptamers; and/or peptides, such asRGD-containing peptides, insulin, EGF, and/or transferrin.

In some embodiments, the targeting ligand is in the form of anN-acetyl-galactosamine trimer. In some embodiments, the targeting ligandis in the form of an N-acetyl-galactosamine tetramer.

Oligomeric Compounds

The targeting ligands disclosed herein can be linked to an oligomericcompound. In some embodiments, the oligomeric compound is anexpression-inhibiting oligomeric compound. In some embodiments, theexpression-inhibiting oligomeric compound is an RNAi agent. In someembodiments, the expression-inhibiting oligomeric compound is adouble-stranded RNAi agent. In some embodiments theexpression-inhibiting oligomeric compound is a single-strandedoligonucleotide. The expression-inhibiting oligomeric compounds may besynthesized using methods commonly used in the art.

The expression-inhibiting oligomeric compounds may include one or moremodified nucleotides. A nucleotide base (or nucleobase) is aheterocyclic pyrimidine or purine compound which is a constituent of allnucleic acids and includes adenine (A), guanine (G), cytosine (C),thymine (T), and uracil (U). As used herein, the term “nucleotide” mayinclude a modified nucleotide or nucleotide mimic, abasic site, or asurrogate replacement moiety. As used herein, a “modified nucleotide” isa nucleotide, nucleotide mimic, abasic site, or a surrogate replacementmoiety other than a ribonucleotide (2′-hydroxyl nucleotide). In someembodiments a modified nucleotide includes a 2′-modified nucleotide(i.e. a nucleotide with a group other than a hydroxyl group at the 2′position of the five-membered sugar ring). Modified nucleotides include,but are not limited to: 2′-modified nucleotides, 2′-O-methyl nucleotides(represented herein as a lower case letter ‘n’ in a nucleotidesequence), 2′-deoxy-2′-fluoro nucleotides (represented herein as Nf,also represented herein as 2′-fluoro nucleotide), 2′-deoxy nucleotides(represented herein as dN), 2′-methoxyethyl (2′-O-2-methoxylethyl)nucleotides, (represented herein as NM or 2′-MOE), 2′-amino nucleotides,2′-alkyl nucleotides, 3′ to 3′ linkages (inverted) nucleotides(represented herein as invdN, invN, invn, invX), non-natural baseincluding nucleotides, locked nucleotides, bridged nucleotides, peptidenucleic acids, 2′,3′-seco nucleotide mimics (unlocked nucleobaseanalogues, represented herein as N_(UNA or) NUNA), locked nucleotide(represented herein as N_(LNA) or NLNA), 3′-O-methoxy (2′internucleotide linked) nucleotide (represented herein as 3′-OMen),2′-F-arabino nucleotides (represented herein as NfANA or Nf_(ANA)),morpholino nucleotides, vinyl phosphonate deoxyribonucleotide(represented herein as vpdN), vinyl phosphonate nucleotides, and abasicnucleotides (represented herein as X or Ab). It is not necessary for allpositions in a given compound to be uniformly modified. Conversely, morethan one modification may be incorporated in a singleexpression-inhibiting oligomeric compound or even in a single nucleotidethereof. The expression-inhibiting oligomeric compounds may besynthesized and/or modified by methods known in the art. Modification ateach nucleotide is independent of modification of the other nucleotides.

Modified nucleobases include synthetic and natural nucleobases, such as5-substituted pyrimidines, 6-azapyrimidines, N-2-, N-6-, andO-6-substituted purines (e.g., 2-amninopropyladenine), 5-propynyluracil,5-propynylcytosine, 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine, 2-thiocytosine,5-halouracil, 5-halocytosine, 5-propynyl uracil, 5-propynyl cytosine,6-azo-uracil, 6-azo-cytosine, 6-azo-thymine, 5-uracil (pseudouracil),4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl andother 8-substituted adenines and guanines, 5-substituted uracils andcytosines (e.g., 5-halo uracils and cytosines (e.g., 5-bromouracil and5-bromnocytosine), 5-trifluoromethyl uracil, 5-trifluoromethylcytosine), 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine,7-deazaguanine, 7-deazaadenine, 3-deazaguanine, and 3-deazaadenine.

For the expression-inhibiting oligomeric compounds described herein, anymodified nucleotides may be linked by phosphate-containing ornon-phosphate-containing covalent internucleoside linkages. Modifiedinternucleoside linkages or backbones include, but are not limited to,5′-phosphorothioate group (represented herein as a lower case ‘s’ beforea nucleotide, as in sN, sn, sNf, or sdN), chiral phosphorothioates,thiophosphate, phosphorodithioates, phosphotriesters,aminoalkyl-phosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkyl-phosphonates, thionoalkylphosphotriesters, morpholinolinkages, boranophosphates having normal 3′-5′ linkages, 2′-5′ linkedanalogs of boranophosphates, and boranophosphates having invertedpolarity wherein the adjacent pairs of nucleoside units are linked 3′-5′to 5′-3′ or 2′-5′ to 5′-2′. In some embodiments, a modifiedinternucleoside linkage or backbone lacks a phosphorus atom. Modifiedinternucleoside linkages lacking a phosphorus atom include, but are notlimited to, short chain alkyl or cycloalkyl inter-sugar linkages, mixedheteroatom and alkyl or cycloalkyl inter-sugar linkages, or one or moreshort chain heteroatomic or heterocyclic inter-sugar linkages. In someembodiments, modified internucleoside backbones include, but are notlimited to, siloxane backbones, sulfide backbones, sulfoxide backbones,sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, alkene-containing backbones,sulfamate backbones, methyleneimino and methylenehydrazino backbones,sulfonate and sulfonamide backbones, amide backbones, and otherbackbones having mixed N, O, S, and CH₂ components.

In some embodiments, an expression-inhibiting oligomeric compound is adouble-stranded RNAi agent, and includes a sense strand and an antisensestrand that are at least partially complementary (at least 70%complementary) to each other. The antisense strand contains a regionhaving a sequence that is perfectly complementary (100% complementary)or at least substantially complementary (at least 85% complementary) toa sequence in a target mRNA. The length of a double-stranded RNAi agentsense strand and antisense strand each can be 16 to 30 nucleotides inlength. The sense and antisense strands can be either the same length orthey can be different lengths. In some embodiments, the sense strand isabout 19 nucleotides in length while the antisense strand is about 21nucleotides in length. In some embodiments, the sense strand is about 21nucleotides in length while the antisense strand is about 23 nucleotidesin length. In other embodiments, the sense and antisense strands areeach independently 17-21 nucleotides in length. In some embodiments,both the sense and antisense strands are each 21-26 nucleotides inlength. In some embodiments, both the sense and antisense strands areeach 26 nucleotides in length. In some embodiments, the sense andantisense strands are each independently 17 to 26 nucleotides in length.In some embodiments, a double-stranded RNAi agent has a duplex length ofabout 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides. This region ofperfect or substantial complementarity between the sense strand and theantisense strand is typically 15-25 (e.g., 15, 16, 17, 18, 19, 20, 21,22, 23, 24, or 25 nucleotides in length) nucleotides in length andoccurs at or near the 5′ end of the antisense strand.

The expression-inhibiting oligomeric compounds that are conjugated tothe ligands disclosed herein optionally and independently include anadditional 1, 2, 3, 4, 5, or 6 nucleotides (as an extension) at the 3′end, the 5′ end, or both the 3′ and 5′ ends of the core sequences. Theseadditional nucleotides, if present, may or may not be complementary tothe corresponding sequence in the targeted mRNA.

In some embodiments, when a double-stranded RNAi agent is conjugated tothe targeting ligands disclosed herein, the additional sense strandadditional nucleotides, if present, may or may not be identical to thecorresponding sequence in the targeted mRNA. The additional antisensestrand additional nucleotides, if present, may or may not becomplementary to the corresponding additional nucleotides of the sensestrand, if present.

Double-stranded RNAi agents can be formed by annealing an antisensestrand with a sense strand.

In some embodiments, the targeting ligand is linked to an RNAi agent atthe 3′ or 5′ end of either the sense strand or the antisense strand ofthe RNAi agent. In some embodiments, the targeting ligand is linked to5′ end of the sense strand. In some embodiments, the targeting ligand islinked to the 3′ end of the sense strand. In some embodiments, thetargeting ligand is linked to the RNAi agent via a labile, cleavable, orreversible bond. In some embodiments, the labile, cleavable, orreversible bond is included in a cleavable moiety added between the RNAiagent and the targeting ligand.

In some embodiments, the expression-inhibiting oligomeric compound is asingle-stranded oligonucleotide. In some embodiments, thesingle-stranded oligonucleotide is utilizes the RNA interferencemechanism to inhibit expression of the target mRNA. In some embodiments,the single-stranded oligonucleotides are active in reducing expressionof the target nucleic acid through a mechanism other than RNAinterference.

In some embodiments, the gene expression level and/or mRNA level of atarget in a subject to whom a described targeting ligand conjugated toan expression-inhibiting oligomeric compound is administered is reducedby at least about 5%, for example, by at least about 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or98% relative to the subject prior to administration or to a subject notreceiving the targeting ligand conjugate. The gene expression leveland/or mRNA level in the subject may be reduced in a cell, group ofcells, and/or tissue of the subject. In some embodiments, the proteinlevel in a subject to whom a described targeting ligand conjugated to anexpression-inhibiting oligomeric compound has been administered isreduced by at least about 5%, for example, by at least about 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 98% relative to the subject prior to being administered thetargeting ligand conjugate or to a subject not receiving the targetingligand conjugate. The protein level in the subject may be reduced in acell, group of cells, tissue, blood, and/or other fluid of the subject.A reduction in gene expression, mRNA, or protein levels can be assessedby any methods known in the art. Reduction or decrease in mRNA leveland/or protein level are collectively referred to herein as inhibiting,decreasing, or reducing the expression of the targeted gene.

Specific expression-inhibiting oligomeric compounds that can be usedwith the targeting ligands disclosed are known in the art. Inparticular, numerous references disclose expression-inhibitingoligomeric compounds that may be conjugated to the targeting ligandsdisclosed herein for delivery of the composition to the liver.Non-limiting examples include U.S. patent application Ser. No.15/281,309, entitled Compositions and Methods for Inhibiting GeneExpression of LPA, which is incorporated herein by reference in itsentirety, discloses various double-stranded expression-inhibitingoligomeric compounds targeting the human apolipoprotein(a) gene [LPA](to inhibit expression of the apo(a) protein which is part of thelipoprotein(a) particle, and thereby the lipoprotein(a) particle(Lp(a))), that are suitable for use with the targeting ligands disclosedherein. The apo(a) gene [LPA] is expressed predominantly in the liver inhumans and non-human primates. Similarly, for example, U.S. patentapplication Ser. No. 15/229,314, entitled RNAi Therapy for Hepatitis BVirus Infection, which is also incorporated herein by reference in itsentirety, discloses various double-stranded expression-inhibitingoligomeric compounds targeting the hepatitis B virus, that are suitablefor use with the targeting ligands disclosed herein. The Hepatitis BVirus is a strict hepatotrophic, double-stranded DNA containing virusand is classified as one member of the Hepadnaviruses, belonging to thefamily of Hepadnaviridae. Further, as another example, U.S. patentapplication Ser. No. 15/229,314, entitled Compositions and Methods forInhibiting Gene Expression of Factor XII, which is incorporated hereinby reference in its entirety, discloses various double-strandedexpression-inhibiting oligomeric compounds targeting the Factor XII (orFactor 12, F12) gene, that are suitable for use with the targetingligands disclosed herein. Factor XII is a serine protease expressedpredominantly in the liver and found in blood. Additionally, as anotherexample U.S. patent application Ser. No. 14/740,307, entitledCompositions and Methods for Inhibiting Gene Expression of Alpha-1AntiTrypsin, which is incorporated herein by reference in its entirety,discloses various double-stranded expression-inhibiting oligomericcompounds targeting the alpha-1 antitrypsin (or AAT) gene, that aresuitable for use with the targeting ligands disclosed herein. AAT is aprotease inhibitor belonging to the serpin superfamily, and normal AATprotein is primarily synthesized in the liver by hepatocytes andsecreted into blood. Further, WO 2016/01123, entitled OrganicCompositions to Treat APOC3-Related Diseases, which is incorporatedherein by reference in its entirety, discloses various double-strandedexpression-inhibiting oligomeric compounds targeting humanapolipoprotein III (APOC3), that are suitable for use with the targetingligands disclosed herein. Apolipoprotein C-III is a constituent oflipoproteins that is believed to inhibit hepatic uptake oftriglyceride-rich particles. Additional references disclosing varioustherapeutic compounds, including expression-inhibiting oligomericcompounds, that may be suitable for use with the targeting ligandsdisclosed herein, can also be found in the art. These include, but arenot limited to, compositions where targeting to the liver would bedesirable.

Pharmaceutical Compositions and Formulations

The targeting ligands disclosed herein, when linked to an oligomericcompound, can be used to treat a subject (e.g., a human or mammal)having a disease or disorder that would benefit from administration ofthe compound. In some embodiments, the targeting ligands disclosedherein, when linked to an expression-inhibiting oligomeric compound, canbe used to treat a subject (e.g., a human) having a disease or disorderthat would benefit from reduction or inhibition in expression of thetarget mRNA. The subject is administered a therapeutically effectiveamount of any one or more expression-inhibiting oligomeric compounds,such as an RNAi agent, that is linked to a targeting ligand disclosedherein. The subject can be a human, patient, or human patient. Thesubject may be an adult, adolescent, child, or infant. The describedpharmaceutical compositions including a targeting ligand linked to anexpression-inhibiting oligomeric compound can be used to provide methodsfor the therapeutic treatment of diseases. Such methods includeadministration of a pharmaceutical composition described herein to ahuman being or animal.

The pharmaceutical compositions and methods disclosed herein maydecrease the level of the target mRNA in a cell, group of cells, groupof cells, tissue, or subject, including: administering to the subject atherapeutically effective amount of a herein describedexpression-inhibiting oligomeric compound that is linked to a targetingligand, thereby inhibiting the expression of a target mRNA in thesubject. In some embodiments, the subject has been previously identifiedas having a pathogenic upregulation of the target gene in the targetedcell or tissue.

In some embodiments, pharmaceutical compositions include at least oneexpression-inhibiting oligomeric compound linked to a targeting ligand.These pharmaceutical compositions are particularly useful in theinhibition of the expression of the target mRNA in a target cell, agroup of cells, a tissue, or an organism. The pharmaceuticalcompositions can be used to treat a subject having a disease or disorderthat would benefit from reduction in the level of the target mRNA, orinhibition in expression of the target gene. The pharmaceuticalcompositions can be used to treat a subject at risk of developing adisease or disorder that would benefit from reduction of the level ofthe target mRNA or an inhibition in expression the target gene. In oneembodiment, the method includes administering a composition including atargeting ligand as described herein linked to an expression-inhibitingoligomeric compound, such as an RNAi agent, to a subject to be treated.In some embodiments, one or more pharmaceutically acceptable excipients(including vehicles, carriers, diluents, and/or delivery polymers) areadded to the pharmaceutical compositions including a targeting ligandlinked to an expression-inhibiting oligomeric compound, thereby forminga pharmaceutical formulation suitable for in vivo delivery to a human.

In some embodiments, the described pharmaceutical compositions includinga targeting ligand linked to an expression-inhibiting oligomericcompound are used for treating or managing clinical presentationsassociated with expression of a target mRNA. In some embodiments, atherapeutically or prophylactically effective amount of one or more ofpharmaceutical compositions is administered to a subject in need of suchtreatment, prevention or management. In some embodiments, administrationof any of the conjugated ligands covalently linked to an oligomericcompound can be used to decrease the number, severity, and/or frequencyof symptoms of a disease in a subject.

The described pharmaceutical compositions including a targeting ligandlinked to an expression-inhibiting oligomeric compound, can be used totreat at least one symptom in a subject having a disease or disorderthat would benefit from reduction or inhibition in expression of atarget mRNA. In some embodiments, the subject is administered atherapeutically effective amount of one or more pharmaceuticalcompositions including an expression-inhibiting oligomeric compound,such as an RNAi agent, linked to a targeting ligand described herein,thereby treating the symptom. In other embodiments, the subject isadministered a prophylactically effective amount of one or more ofexpression-inhibiting oligomeric compounds thereby preventing the atleast one symptom.

In some embodiments, the expression or level of a target mRNA in asubject to whom an expression-inhibiting oligomeric compound linked to atargeting ligand disclosed herein is administered is reduced by at leastabout 5%, for example, but at least about 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%relative to the subject not receiving the pharmaceutical composition.The gene expression level in the subject may be reduced in a cell, groupof cells, and/or tissue of the subject. In some embodiments, the levelof mRNA is reduced. In other embodiments, the expressed protein level isreduced. In some embodiments, the level of protein in a subject to whoman expression-inhibiting oligomeric compound linked to a targetingligand disclosed herein is administered is reduced by at least about 5%,for example, but at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% relative to thesubject not receiving the pharmaceutical composition. Reduction inexpression, mRNA levels, or protein levels can be assessed by anymethods known in the art. Reduction or decrease in mRNA level and/orprotein level are collectively referred to herein as a reduction ordecrease in target RNA or inhibiting or reducing the expression oftarget mRNA.

The route of administration is the path by which anexpression-inhibiting oligomeric compound is brought into contact withthe body. In general, methods of administering drugs and nucleic acidsfor treatment of a mammal are well known in the art and can be appliedto administration of the compositions described herein. Theexpression-inhibiting oligomeric compound linked to the herein describedtargeting ligands can be administered via any suitable route in apreparation appropriately tailored to the particular route. Thus, hereindescribed pharmaceutical compositions can be administered by injection,for example, intravenously, intramuscularly, intracutaneously,subcutaneously, intraarticularly, or intraperitoneally. In someembodiments, there herein described pharmaceutical compositions and beadministered via inhalation.

The pharmaceutical compositions including an expression-inhibitingoligomeric compound linked to a targeting ligand described herein can bedelivered to a cell, group of cells, tumor, tissue, or subject usingoligonucleotide delivery technologies known in the art. In general, anysuitable method recognized in the art for delivering a nucleic acidmolecule (in vitro or in vivo) can be adapted for use with a hereindescribed compositions. For example, delivery can be by localadministration, (e.g., direct injection, implantation, or topicaladministering), systemic administration, or subcutaneous, intravenous,intraperitoneal, or parenteral routes, including intracranial (e.g.,intraventricular, intraparenchymal and intrathecal), intramuscular,transdermal, airway (aerosol), nasal, oral, rectal, or topical(including buccal and sublingual) administration. In certainembodiments, the compositions are administered by subcutaneous orintravenous infusion or injection.

Accordingly, in some embodiments, the herein described pharmaceuticalcompositions may comprise one or more pharmaceutically acceptableexcipients. In some embodiments, the pharmaceutical compositionsdescribed herein can be formulated for administration to a subject.

As used herein, a pharmaceutical composition or medicament includes apharmacologically effective amount of at least one of the describedtherapeutic compounds and one or more pharmaceutically acceptableexcipients. Pharmaceutically acceptable excipients (excipients) aresubstances other than the Active Pharmaceutical ingredient (API,therapeutic product, e.g., F12 RNAi agent) that are intentionallyincluded in the drug delivery system. Excipients do not exert or are notintended to exert a therapeutic effect at the intended dosage.Excipients may act to a) aid in processing of the drug delivery systemduring manufacture, b) protect, support or enhance stability,bioavailability or patient acceptability of the API, c) assist inproduct identification, and/or d) enhance any other attribute of theoverall safety, effectiveness, of delivery of the API during storage oruse. A pharmaceutically acceptable excipient may or may not be an inertsubstance.

Excipients include, but are not limited to: absorption enhancers,anti-adherents, anti-foaming agents, anti-oxidants, binders, bufferingagents, carriers, coating agents, colors, delivery enhancers, deliverypolymers, dextran, dextrose, diluents, disintegrants, emulsifiers,extenders, fillers, flavors, glidants, humectants, lubricants, oils,polymers, preservatives, saline, salts, solvents, sugars, suspendingagents, sustained release matrices, sweeteners, thickening agents,tonicity agents, vehicles, water-repelling agents, and wetting agents.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). Itshould be stable under the conditions of manufacture and storage andshould be preserved against the contaminating action of microorganismssuch as bacteria and fungi. The carrier can be a solvent or dispersionmedium containing, for example, water, ethanol, polyol (for example,glycerol, propylene glycol, and liquid polyethylene glycol), andsuitable mixtures thereof. The proper fluidity can be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. In many cases, it will be preferable to include isotonicagents, for example, sugars, polyalcohols such as mannitol, sorbitol,and sodium chloride in the composition. Prolonged absorption of theinjectable compositions can be brought about by including in thecomposition an agent which delays absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfilter sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation include vacuumdrying and freeze-drying which yields a powder of the active ingredientplus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Formulations suitable for intra-articular administration can be in theform of a sterile aqueous preparation of the drug that can be inmicrocrystalline form, for example, in the form of an aqueousmicrocrystalline suspension. Liposomal formulations or biodegradablepolymer systems can also be used to present the drug for bothintra-articular and ophthalmic administration.

Formulations suitable for topical administration, including eyetreatment, include liquid or semi-liquid preparations such as liniments,lotions, gels, applicants, oil-in-water or water-in-oil emulsions suchas creams, ointments or pastes; or solutions or suspensions such asdrops. Formulations for topical administration to the skin surface canbe prepared by dispersing the drug with a dermatologically acceptablecarrier such as a lotion, cream, ointment or soap. Useful are carrierscapable of forming a film or layer over the skin to localize applicationand inhibit removal. For topical administration to internal tissuesurfaces, the agent can be dispersed in a liquid tissue adhesive orother substance known to enhance adsorption to a tissue surface. Forexample, hydroxypropylcellulose or fibrinogen/thrombin solutions can beused to advantage. Alternatively, tissue-coating solutions, such aspectin-containing formulations can be used.

For inhalation treatments, inhalation of powder (self-propelling orspray formulations) dispensed with a spray can, a nebulizer, or anatomizer can be used. Such formulations can be in the form of a finepowder for pulmonary administration from a powder inhalation device orself-propelling powder-dispensing formulations. In the case ofself-propelling solution and spray formulations, the effect can beachieved either by choice of a valve having the desired spraycharacteristics (i.e., being capable of producing a spray having thedesired particle size) or by incorporating the active ingredient as asuspended powder in controlled particle size. For administration byinhalation, the compounds also can be delivered in the form of anaerosol spray from pressured container or dispenser which contains asuitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

Systemic administration also can be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants generally are known in the art, and include, forexample, for transmucosal administration, detergents and bile salts.Transmucosal administration can be accomplished through the use of nasalsprays or suppositories. For transdermal administration, the activecompounds typically are formulated into ointments, salves, gels, orcreams as generally known in the art.

The active compounds can be prepared with carriers that will protect thecompound against rapid elimination from the body, such as a controlledrelease formulation, including implants and microencapsulated deliverysystems. Biodegradable, biocompatible polymers can be used, such asethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Methods for preparation of suchformulations will be apparent to those skilled in the art. Liposomalsuspensions can also be used as pharmaceutically acceptable carriers.These can be prepared according to methods known to those skilled in theart, for example, as described in U.S. Pat. No. 4,522,811.

Oral or parenteral compositions can be formulated in dosage unit formfor ease of administration and uniformity of dosage. Dosage unit formrefers to physically discrete units suited as unitary dosages for thesubject to be treated; each unit containing a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the disclosure are dictated by and directlydependent on the unique characteristics of the active compound and thetherapeutic effect to be achieved, and the limitations inherent in theart of compounding such an active compound for the treatment ofindividuals. Furthermore, administration can be by periodic injectionsof a bolus, or can be made more continuous by intravenous, intramuscularor intraperitoneal administration from an external reservoir (e.g., anintravenous bag).

In conjunction with the methods of the present disclosure,pharmacogenomics (i.e., the study of the relationship between anindividual's genotype and that individual's response to a foreigncompound or drug) can be considered. Differences in metabolism oftherapeutics can lead to severe toxicity or therapeutic failure byaltering the relation between dose and blood concentration of thepharmacologically active drug. Thus, a physician or clinician canconsider applying knowledge obtained in relevant pharmacogenomicsstudies in determining whether to administer a drug as well as tailoringthe dosage and/or therapeutic regimen of treatment with the drug.

A pharmaceutical composition can contain other additional componentscommonly found in pharmaceutical compositions. Such additionalcomponents include, but are not limited to: anti-pruritics, astringents,local anesthetics, or anti-inflammatory agents (e.g., antihistamine,diphenhydramine, etc.). It is also envisioned that cells, tissues orisolated organs that express or comprise the herein defined RNAi agentsmay be used as “pharmaceutical compositions.” As used herein,“pharmacologically effective amount,” “therapeutically effectiveamount,” or simply “effective amount” refers to that amount of an RNAiagent to produce a pharmacological, therapeutic or preventive result.

Generally, an effective amount of an active compound will be in therange of from about 0.1 to about 100 mg/kg of body weight/day, e.g.,from about 1.0 to about 50 mg/kg of body weight/day. In someembodiments, an effective amount of an active compound will be in therange of from about 0.25 to about 5 mg/kg of body weight per dose. Insome embodiments, an effective amount of an active ingredient will be inthe range of from about 0.5 to about 3 mg/kg of body weight per dose.The amount administered will also likely depend on such variables as theoverall health status of the patient, the relative biological efficacyof the compound delivered, the formulation of the drug, the presence andtypes of excipients in the formulation, and the route of administration.Also, it is to be understood that the initial dosage administered can beincreased beyond the above upper level in order to rapidly achieve thedesired blood-level or tissue level, or the initial dosage can besmaller than the optimum.

For treatment of disease or for formation of a medicament or compositionfor treatment of a disease, the pharmaceutical compositions describedherein including an expression-inhibiting oligomeric compound, such asan RNAi agent, linked to a targeting ligand, can be combined with anexcipient or with a second therapeutic agent or treatment including, butnot limited to: a second or other expression-inhibiting oligomericcompound, a small molecule drug, an antibody, an antibody fragment,and/or a vaccine.

The described targeting ligands, when linked to expression-inhibitingoligomeric compounds, and when added to pharmaceutically acceptableexcipients or adjuvants, can be packaged into kits, containers, packs,or dispensers. The pharmaceutical compositions described herein may bepackaged in pre-filled syringes or vials.

The above provided embodiments are now illustrated with the following,non-limiting examples.

EXAMPLES

The following examples are not limiting and are intended to illustratecertain embodiments disclosed herein.

Some of the abbreviations used in the following experimental details ofthe synthesis of the examples are defined below: h or hr=hour(s);min=minute(s); mol=mole(s); mmol=millimole(s); M=molar; μM=micromolar;g=gram(s); μg=microgram(s); rt or RT=room temperature; L=liter(s);mL=milliliter(s); wt=weight; Et₂O=diethyl ether; THF=tetrahydrofuran;DMSO=dimethyl sulfoxide; EtOAc=ethyl acetate; Et₃N or TEa=triethylamine;i-Pr₂NEt or DIPEA or DIEA=diisopropylethylamine; CH₂Cl₂ or DCM=methylenechloride; CHCl₃=chloroform; CDCl₃=deuterated chloroform; CCl₄=carbontetrachloride; MeOH=methanol; EtOH=ethanol; DMF=dimethylformamide;BOC=t-butoxycarbonyl; CBZ=benzyloxycarbonyl; TBS=t-butyldimethylsilyl;TBSCl=t-butyldimethylsilyl chloride; TFA=trifluoroacetic acid;DMAP=4-dimethylaminopyridine; NaN₃=sodium azide; Na₂SO₄=sodium sulfate;NaHCO₃=sodium bicarbonate; NaOH=sodium hydroxide; MgSO₄=magnesiumsulfate; K₂CO₃=potassium carbonate; KOH=potassium hydroxide;NH₄OH=ammonium hydroxide; NH₄Cl=ammonium chloride; SiO₂=silica;Pd—C=palladium on carbon; HCl=hydrogen chloride or hydrochloric acid;NMM=N-methylmorpholine; H₂=hydrogen gas; KF=potassium fluoride;EDC-HCl═N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride;MTBE=methyl-tert-butyl ether; MeOH=methanol; Ar=argon; SiO₂=silica;R_(T)=retention time.

Additionally, exemplary expression-inhibiting oligomeric compoundssuitable for use with the targeting ligands disclosed herein are setforth in various Tables in the Examples that follow. The followingnotations are used to indicate modified nucleotides for sequences setforth in the Tables disclosed herein:

-   -   N=2′-OH (unmodified) ribonucleotide (capital letter without for        d indication)    -   n=2′-OMe modified nucleotide    -   Nf=2′-fluoro modified nucleotide    -   dN=2′-deoxy nucleotides    -   N_(UNA)=2′,3′-seco nucleotide mimics (unlocked nucleobase        analogs)    -   N_(LNA)=locked nucleotide    -   Nf_(ANA)=2′-F-Arabino nucleotide    -   NM=2′-methoxyethyl nucleotide    -   X or Ab=abasic ribose    -   R=ribitol    -   (invdN)=inverted deoxyribonucleotide (3′-3′ linked nucleotide)    -   (invAb)=inverted abasic nucleotide    -   (invX)=inverted abasic nucleotide    -   (invn)=inverted 2′-OMe nucleotide    -   s=phosphorothioate linked nucleotide    -   vpdN=vinyl phosphonate deoxyribonucleotide    -   (3′OMen)=3′-OMe nucleotide    -   (5Me-Nf)=5′-Me, 2′-fluoro nucleotide    -   cPrp=cyclopropyl phosphonate

The compounds of the present disclosure can be made using syntheticchemical techniques known to those of skill in the art.

Example 1. Synthesis of Targeting Ligand Phosphoramidite CompoundStructure 101b 1) Preparation of Tri-tert-butylN—[N-(Benzyloxycarbonyl)-L-γ-glutamyl]-L-glutamate (3)

To a nitrogen-flushed, 250-mL 3-neck round-bottomed flask equipped witha thermocouple, magnetic stir bar, nitrogen inlet, and powder funnel wasadded 1 (10.00 g, 29.64 mmol) followed by THF (100 mL). The resultingsolution was stirred, and N-methylmorpholine (7.82 mL, 71.15 mmol) wasadded.

The powder funnel was replaced with a rubber septum, and the mixture wascooled using an ice bath to 0° C. Isobutyl chloroformate (iBuCOCl, 3.85mL, 29.64 mmol, 1.0 equivalents) was added to the reaction mixturedropwise over 10 minutes, maintaining a pot temperature of less than4.0° C. Following addition, the mixture was stirred 40 minutes more, andthe septum was replaced with a powder funnel. To the reaction mixturewas added 2 (8.767 g, 29.64 mmol, 1.0 equivalents) portion-wise over 15minutes, maintaining a pot temperature of less than 4.0° C. Followingaddition of 2, the ice bath and powder funnel were removed, and thereaction was allowed to warm to ambient temperature over the course ofthe remaining steps. The clear, colorless solution was aged 25 minutesfollowing the addition of 2.

A sample of the reaction (98 μL diluted into 5.0 mL ACN in a 5-mLvolumetric flask) was taken 40 minutes after the start of addition of 2and analyzed for percent conversion by RP-HPLC. There was found to be23% remaining of 1, so after 60 minutes of reaction, additional iBuCOCl(1.16 mL, 30 mol %) and 2 (2.63 g, 30 mol %) were added sequentially.The solution was aged for an additional 60 minutes, until a sampleshowed greater than 99% conversion by HPLC. Total reaction time was 2.5hours from the start of the initial addition of 2.

The reaction solution was poured into a stirring solution of 0.5 MHCl_((aq)) chilled in an ice bath to 3° C. and stirred about 5 minutes.The quenched reaction mixture was transferred to a 500-mL separatoryfunnel, and ethyl acetate (100 mL) was added. The layers were separated,and the organic phase was washed with brine (100 mL), dried over MgSO₄,filtered into a 500-mL round-bottomed flask, and concentrated in vacuo,affording a thick colorless oil. The oil was dissolved in MTBE (100 mL)and concentrated in vacuo, once again yielding a thick colorless oil.

To the stirring oil was added hexanes (100 mL). White haze appeared inthe solution, which then disappeared upon further stirring. Seedcrystals were added, and the mixture was allowed to stir for 40 minutes,during which time white crystals slowly formed.

Within 20 minutes, the slurry was thick enough to impede stirring, andadditional hexanes (50 mL) was added. After 40 minutes, the slurry wasfiltered over a coarse fritted funnel, washed three times with hexanes(˜10 mL each), and air-dried in the funnel for 1 hour, affording 3 as afine white powder (15.64 g, 91%). ¹H NMR of compound 3 is shown atFIG. 1. On 75 gram scale the yield was 917% with purity 99%.

2) Preparation of N—[N-(Benzyloxycarbonyl)-L-γ-glutamyl]-L-glutamic Acid(4)

To a 3000-mL, 3-necked round-bottomed flask equipped with an overheadstirrer, powder funnel, thermocouple, and heating mantle was added 3(72.57 g, 125.4 mmol) and formic acid (reagent grade, >95%, 1.45 L, 20vol. equiv.). The powder funnel was replaced by a stopper/N₂, and theresulting solution was heated to 45° C. and stirred for 1 hour, withmonitoring by RP-HPLC. The reaction was deemed complete when less than2.0 area % of mono-t-butyl esters remained.

A sample of the reaction (50 μL diluted into 950 μL of H₂O) was taken 60minutes after the addition of formic acid, and the sample was analyzedby RP-HPLC for the percent of mono-t-butyl esters remaining. Theanalysis showed that 1.8% mono-t-Bu esters remained; therefore, at 90minutes, the heat was removed.

The reaction was diluted with toluene and acetonitrile (ACN, 1500 mLeach), and the mixture was concentrated in vacuo. Formic acid wasazeotropically removed with 1:1 ACN:toluene (˜600 mL), and twice withACN (˜500 mL each). The material was dried on high vacuum overnight toafford a white foamy solid compound 4 (54.3 g, quantitative yield). ¹HNMR of compound 4 (L/N 1321-063B) is shown at FIG. 2.

3) Preparation of N—[N-(Benzyloxycarbonyl)-L-γ-glutamyl]-L-glutamicAcid, tri-[NAG-PEG₂]-amide (6)

To a 1-liter round-bottomed flask was added NAG-amine p-tosylate salt(5, 59.19 g, 97.6 mmol, 4.13 equiv.) and Z-bis-Glu triacid (4, 10.01 g,23.6 mmol, 1.0 equiv.). The mixture was dissolved in acetonitrile (500mL) and concentrated in vacuo to remove water azeotropically. Theresidue was dissolved in fresh acetonitrile (400 mL) and transferred toa nitrogen-flushed 1-liter 3-neck round-bottomed flask containing a stirbar and equipped with a thermocouple. Water content was measured by KF(257 ppm).

To the stirring solution under nitrogen was added TBTU (28.20 g, 87.8mmol, 3.7 equiv.) via a powder funnel. Residual TBTU on the funnel wasrinsed into the reaction using additional acetonitrile (100 mL). DIPEA(34.0 mL, 25.2 g, 8.0 equiv.) was added dropwise via syringe over 20minutes, maintaining a reaction temperature below 25° C. The mixture wasstirred for 2 hours from the start of DIPEA addition, with monitoring byHPLC. Analysis at 78 minutes showed complete consumption of startingmaterial.

After two hours, the solvent was removed in vacuo. The resulting thickoil was dissolved in dichloromethane (1000 mL) and washed with 1.0 NHCl_((aq)) (3×500 mL) and saturated NaHCO_(3(aq)) (3×500 mL). Theorganic layer was dried over Na₂SO₄, filtered, and concentrated in vacuoto afford an off-white waxy solid (33.5 g).

Flash column chromatography was performed on an ISCO CombiFlashautomated purification system using chloroform and methanol as eluents.All fractions suspected to contain product based on the UV chromatogram(220 nm) were analyzed by HPLC, and all fractions containing at least97.0% AUC of product were pooled and concentrated to afford 18.75 g(97.0% purity) of 6. Impure fractions were pooled to yield an additional12.2 g (78.8% purity) of 6. Total yield of 6 was 70.9%. ¹H NMR ofcompound 6 is shown at FIG. 3.

4) Preparation of Tri-NAG-bis-Glu-NH₂ Tosylate Salt (7)

Compound 6 (5.737 g, 3.46 mmol) in MeOH (155 mL) with p-TsOH—H₂O (0.657g, 3.46 mmol) was hydrogenated in presence of Pd/C 10% (688 mg) for 6 h.TLC (CHCl₃; MeOH=8.5:1.5) confirmed that the reaction was completed bythat time. The reaction flask was filled with Ar, EtOH was added (200mL) and the solution was filtered through celite cake. The product wasconcentrated and dried in vacuo. Yield 4.81 g product tosylate salt 7.¹H NMR of compound 7 is shown at FIG. 4.

5) Preparation of Tri-NAG-bis-Glu-NH-PEG₆-OH (9)

Procedure A (if Tri-NAG Amine Salt 7 is Less than 96% Pure):

NAG amine salt 7 (˜90% pure, 18.50 g, 10.90 mmol) and HO-PEG₆-CO₂TFPester 8 (6.57 g, 13.08 mmol) were dissolved in dichloromethane (185 mL)and cooled to 0° C. To this solution was added triethylamine (6.10 mL,43.59 mmol). The solution was allowed to warm to room temperature andstirred for 18 hours with monitoring by HPLC. The reaction was quenchedwith saturated aqueous NaHCO₃ and brine (1:1, 140 mL), stirred for 30min at RT, and the layers were separated. The organic layer was washedwith saturated aqueous NaHCO₃ (3×140 mL) and brine (1:1) and dried withNa₂SO₄. The drying agent was filtered and the solution was concentratedand purified via flash chromatography, which gave 9 (13.56 g, 67%) as awhite solid material. ¹H NMR of compound 9 is shown at FIG. 5.

Flash column chromatography was performed on an ISCO CombiFlashautomated purification system using dichloromethane and methanol aseluents. Pure fractions were pooled and concentrated to afford 13.56 gof 9 (99% purity). Impure fractions were pooled to yield 4.9 g of 9(˜95% purity).

Procedure B (if Tri-NAG Amine Salt 7 is Greater than 96% Pure):

Product 7 (1.94 g, 1.272 mmol) in DCM (40 mL) was stirred under Ar withHO-PEG₆-CO₂TFP ester 8 (767 mg, 1.526 mmol) and DIPEA (443 μL, 2.544mmol) for 16 h. The reaction mixture was concentrated in vacuo,dissolved in CHCl₃ and added dropwise to stirring Et₂O (90 mL). Theprecipitate was separated, rinsed with Et₂O (3×35 mL) and dried invacuo. Yield 2.275 g (96%).

6) Preparation of Tri-NAG-bis-Glu-NH-PEG₆ Phosphoramidite (10)

Compound 9 (6.62 g, 3.56 mmol) and 4,5-dicyanoimidazole (0.11 g, 0.89mmol) were dissolved in anhydrous dichloromethane (230 mL) and placedunder nitrogen atmosphere. To this mixture, a solution of2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (“Phos reagent”,1.46 mL, 4.62 mmol) in anhydrous dichloromethane (5 mL) was addeddropwise over 5 minutes. The reaction mixture was stirred at roomtemperature for 3 h with monitoring HPLC (<1% SM remaining).

The reaction mixture was washed with saturated aqueous NaHCO₃ (2×150mL), 3% DMF in H₂O (v/v, 2×150 mL), H₂O (3×150 mL), and brine (1×150mL), and the organic layer was dried with Na₂SO₄. The drying agent wasfiltered, and the solution was concentrated in vacuo to give crudeproduct. Crude product was suspended in 5% toluene-hexane (50 mL) andstirred for 5 minutes, after which the solvent was decanted. The processwas repeated with 5% toluene-hexane (1×50 mL) and hexane (2×50 mL). Thesolids were dried over vacuum resulting 6.69 g 10 as a white solidmaterial (91%) (compound 10). ¹H NMR of compound 10 (Structure 101dherein) is shown at FIG. 6.

Example 2. Synthesis of Targeting Ligand Phosphoramidite CompoundStructure 103d 1) Preparation of Tri-NAG-bis-Glu-NH-PEG₄-OH (12)

Product 7 (2.44 g, 1.44 mmol), from Example 1 above, was dissolved inDCM (30 mL) and placed under argon atmosphere. To the solution was addedHO-PEG₄-CO₂TFP ester 11 (717 mg, 1.73 mmol) and DIPEA (502 μL, 2.88mmol). The resulting mixture was stirred for 16 h. The reaction mixturewas concentrated in vacuo and redissolved in CHCl₃. The solution wasthen added dropwise to stirring Et₂O (90 mL). The precipitate wasseparated, rinsed with Et₂O and dried in vacuo to yield 2.60 g (102%) ofproduct 12 that was used without further purification.

2) Preparation of Tri-NAG-bis-Glu-NH-PEG₄ Phosphoramidite (13)

Product 12 (1.80 g, 1.01 mmol) was coevaporated with pyridine twicebefore being dissolved in anhydrous dichloromethane (25 mL) and placedunder argon atmosphere. To the solution was added diisopropylammoniumtetrazolide (87 mg, 0.51 mmol) and2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (458 mg, 1.52mmol). The reaction mixture was stirred at room temperature for 5 h withmonitoring by TLC (CHCl₃:MeOH:Et₃N 95:5:2). Once all starting materialhad been consumed, the reaction mixture was diluted with DCM (250 mL)and washed with saturated aqueous NaHCO₃ (100 mL) and saturated aqueousbrine (100 mL). The organic layer was dried with sodium sulfate,filtered, and concentrated. The crude was purified by columnchromatography (DCM:MeOH:Et₃N 97:3:2) to yield 1.04 g (53%) of compound13. ¹H NMR of compound 13 (Structure 103d herein) is shown at FIG. 7.

Example 3. Synthesis of Targeting Ligand Phosphoramidite CompoundStructure 102d 1) Preparation of Tri-NAG-bis-Glu-NH-PEG₈-OH (15)

Product 7 (3.09 g, 1.82 mmol), from Example 1 above, was dissolved inDCM (30 mL) and placed under argon atmosphere. To the solution was addedHO-PEG₈-CO₂TFP ester 14 (1.29 g, 2.18 mmol) and DIPEA (634 μL, 3.64mmol). The resulting mixture was stirred for 16 h. The reaction mixturewas concentrated in vacuo and redissolved CHCl₃. The solution was thenadded dropwise to stirring Et₂O (180 mL). The precipitate was separated,rinsed with Et₂O and dried in vacuo to yield 3.54 g (99%) of product 15that was used without further purification.

2) Preparation of Tri-NAG-bis-Glu-NH-PEG₈ Phosphoramidite (16)

Product 15 (1.79 g, 0.92 mmol) was coevaporated with pyridine twicebefore being dissolved in anhydrous dichloromethane (25 mL) and placedunder argon atmosphere. To the solution was added diisopropylammoniumtetrazolide (79 mg, 0.46 mmol) and2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (416 mg, 1.38mmol). The reaction mixture was stirred at room temperature for 3 h withmonitoring by TLC (CHCl₃:MeOH:Et₃N 95:5:2). Once all starting materialhad been consumed, the reaction mixture was concentrated in vacuo andredissolved in DCM. The solution was then added dropwise to stirringEt₂O (90 mL). The precipitate was separated, rinsed with Et₂O, anddried. The crude was purified by column chromatography (CHCl₃:MeOH:Et₃N97:3:2) to yield 950 mg (48%) of compound 16. ¹H NMR of compound 16(Structure 102d herein) is shown at FIG. 8.

Example 4. Oligonucleotide Composition Synthesis

A. Synthesis.

RNAi agents was synthesized according to phosphoramidite technology onsolid phase used in oligonucleotide synthesis. Depending on the scale,either a MerMade96E® (Bioautomation) or a MerMade12® (Bioautomation) wasused. Syntheses were performed on a solid support made of controlledpore glass (CPG, 500 Å or 600 Å, obtained from Prime Synthesis, Aston,Pa., USA). All RNA and 2′-modified RNA phosphoramidites were purchasedfrom Thermo Fisher Scientific (Milwaukee, Wis., USA). Specifically, thefollowing 2′-O-methyl phosphoramidites were used:(5′-O-dimethoxytrityl-N⁶-(benzoyl)-2′-O-methyl-adenosine-3′-O-(2-cyanoethyl-N,N-diisopropy-lamino)phosphoramidite,5′-O-dimethoxy-trityl-N⁴-(acetyl)-2′-O-methyl-cytidine-3′-O-(2-cyanoethyl-N,N-diisopropyl-amino)phosphoramidite,(5′-O-dimethoxytrityl-N²-(isobutyryl)-2′-O-methyl-guanosine-3′-O-(2-cyano-ethyl-N,N-diisopropylamino)phosphoramidite,and5′-O-dimethoxy-trityl-2′-O-methyl-uridine-3′-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite.The 2′-deoxy-2′-fluoro-phosphoramidites carried the same protectinggroups as the 2′-O-methyl RNA amidites. Targeting ligand containingphosphoramidites were dissolved in anhydrous dichloromethane oranhydrous acetonitrile (50 mM), while all other amidites were dissolvedin anhydrous acetonitrile (50 mM) and molecular sieves (3 Å) were added.5-Benzylthio-1H-tetrazole (BTT, 250 mM in acetonitrile) or5-Ethylthio-1H-tetrazole (ETT, 250 mM in acetonitrile) was used asactivator solution. Coupling times were 10 min (RNA), 15 min (targetingligand), 90 sec (2′OMe), and 60 sec (2′F). In order to introducephosphorothioate linkages, a 100 mM solution of 3-phenyl1,2,4-dithiazoline-5-one (POS, obtained from PolyOrg, Inc., Leominster,Mass., USA) in anhydrous Acetonitrile was employed.

B. Cleavage and Deprotection of Support Bound Oligomer.

After finalization of the solid phase synthesis, the dried solid supportwas treated with a 1:1 volume solution of 40 wt. % methylamine in waterand 28% ammonium hydroxide solution (Aldrich) for two hours at 30° C.The solution was evaporated and the solid residue was reconstituted inwater (see below).

C. Purification.

Crude oligomers were purified by anionic exchange HPLC using a TKSgelSuperQ-5PW 13 u column and Shimadzu LC-8 system. Buffer A was 20 mMTris, 5 mM EDTA, pH 9.0 and contained 20% Acetonitrile and buffer B wasthe same as buffer A with the addition of 1.5 M sodium chloride. UVtraces at 260 nm were recorded. Appropriate fractions were pooled thenrun on size exclusion HPLC using a GE Healthcare XK 16/40 column packedwith Sephadex G-25 medium with a running buffer of 100 mM ammoniumbicarbonate, pH 6.7 and 20% Acetonitrile.

D. Annealing.

Complementary strands were mixed by combining equimolar RNA solutions(sense and antisense) in 0.2×PBS (Phosphate-Buffered Saline, 1×,Corning, Cellgro) to form the RNAi agents. This solution was placed intoa thermomixer at 70° C., heated to 95° C., held at 95° C. for 5 min, andcooled to room temperature slowly. Some RNAi agents were lyophilized andstored at −15 to −25° C. Duplex concentration was determined bymeasuring the solution absorbance on a UV-Vis spectrometer in 0.2×PBS.The solution absorbance at 260 nm was then multiplied by a conversionfactor and the dilution factor to determine the duplex concentration.Unless otherwise stated, all conversion factor was 0.037 mg/(mL·cm). Forsome experiments, a conversion factor was calculated from anexperimentally determined extinction coefficient.

Example 5. Properties of Phosphoramidite-Containing Compounds thatInclude Targeting Ligands with Varying Lengths of PEG Linkers

The following targeting ligand phosphoramidite compounds weresynthesized according to the methods disclosed above in Examples 1-4:

Each of the phosphoramidite compounds of Structure 101d, 102d, and 103d,was delivered at 16 equivalents for conjugation at the 5′ end of thesingle stranded oligonucleotide AM03704-SS, which is a sense strand thatmay be used in synthesizing a double-stranded RNAi agent targeting F12.AM03704 has the nucleotide sequence shown in the Table below:

Table 1. Sense Strand Sequence of Example 5.

TABLE 1 Sense Strand Sequence of Example 5. 5′ → 3′ SEQ ID NO:Sense Strand Sequence: uauaugscsccaagaAfaGfugaaagacc(invdA) 1(AM03704-SS)

The compositions were solubilized in dichloromethane (DCM) and driedover sieves. The phosphoramidite compound of Structure 103d (i.e.,having a PEG-4 linker) presented gelling issues at both 0.05M and 0.25M.As shown in FIG. 9, under these conditions only a very small amount ofthe targeting ligand Structure 103d was able to conjugate to 5′ terminalend of oligonucleotide AM03704-SS.

Structures 101d and 102d both showed conjugation of the targeting ligandto the oligonucleotide. FIG. 9 shows the HPLC chromatograph for AM03704conjugated to Structure 101d. It was determined that for the targetingligand of Structure 101, approximately 78% of the targetingligand-conjugated oligonucleotide (FLP=full length product) was formed.FIG. 10 shows the HPLC chromatograph for AM03704 conjugated to Structure102d. Approximately 40% of the targeting ligand-conjugatedoligonucleotide was formed, while approximately 60% of theoligonucleotide remained unconjugated.

Surprisingly and unexpectedly, at 16 equivalents, Structure 101dsubstantially outperformed both Structure 102d and Structure 103d withrespect to conjugation to the oligonucleotide at the 5′ end of thesequence. Additionally, both Structure 101d and 102d showed greatersolubility compared to Structure 103d. As noted above, Structure 103dwas difficult to dissolve using standard concentrations and solventconditions typical for oligonucleotide synthesis. The manufacture of thetargeting ligands linked to expression-inhibiting oligomeric compoundshaving the targeting ligand of Structure 103 (by using thephosphoramidite compound of Structure 103d) required the addition ofmore aggressive polar solvents.

Example 6. Comparison of 3′ and 5′ Sense Strand Attachment Sites forGalNAc Targeting Ligands Using F12 Expression-Inhibiting OligomericCompounds in Wild Type Mice

To assess differences in the site of attachment of GalNAc ligandsbetween the 3′ and 5′ terminal end of the sense strand,expression-inhibiting oligomeric compounds (double-stranded RNAi agents)directed to F12 (referred to as F12 RNAi agents herein) were preparedhaving the sequences set forth in the following Table 2:

TABLE2F12 expression-inhibiting oligomeric compounds (RNAi agent duplexes) ofExample 6. SEQ Duplex ID: AD02803 5′→3′ ID NO: Sense Strand Sequence:uAuAugscsccaagaAfaGfugaaagacca(NAG15) 2 (AM03628-SS)Antisense Strand Sequence: usGfsgucuuUfcAfcuuUfcuugggcsuscuAu 3(AM03157-AS) SEQ Duplex ID: AD02807 5′→3′ ID NO: Sense Strand Sequence:(NAG18)uauaugscsccaagaAfaGfugaaagacc(invdA) 4 (AM03632-SS)Antisense Strand Sequence: usGfsgucuuUfcAfcuuUfcuugggcsuscuAu 5(AM03157-AS)

In Table 2, above, the following notations are used:

(NAG18) has the chemical structure represented by Structure 2 herein.

Each strand of the F12 RNAi agents was synthesized according tophosphoramidite technology on solid phase used in oligonucleotidesynthesis using either a MerMade96E® (Bioautomation) or a MerMade12®(Bioautomation), and complementary strands were mixed by combiningequimolar RNA solutions (sense and antisense) in 0.2×PBS(Phosphate-Buffered Saline, 1×, Corning, Cellgro) to form the duplexes,following the methods generally described in Example 4 herein.

The F12 RNAi agents linked to the respective GalNAc ligand (i.e.,(NAG15) or (NAG18)) were combined in a pharmaceutically acceptablebuffer as known in the art for subcutaneous (SC) injection.

The F12 RNAi agents linked to the respective GalNAc ligands weredelivered via SC injection. On day 1, SC injection was made into theloose skin on the back between the shoulders of 200 μl solution/20 gmouse containing either saline or a 3 mg/kg (mpk) dose of one of two F12RNAi agents (AD02803 or AD02807) in buffered saline. There were three(3) wild type mice per treatment group. As shown above, AD02803 includes(NAG15) attached to the 3′ terminal end of the sense strand, while AD2807 includes (NAG18) attached to the 5′ end of the sense strand.

Serum samples from treated mice were taken on days 8, 15, 22 and 29 tomonitor knockdown. Knockdown was measured by quantifying circulatingmouse F12 protein (mF12) levels in serum by an internally developed mF12alphaLISA® (Perkin Elmer). Expression at a specific bleed date wasnormalized to the mean of the saline control group for that same date.

FIG. 12 shows the results from this study. At nadir (day 22), AD02803showed approximately 70% reduction in circulating F12 levels, whileAD02807 showed a greater than 80% reduction. The data also show adifference in length of knockdown effect, as at day 29 AD02803-treatedmice showed a faster return to baseline as compared to AD2807-treatedmice. These data support that the linkage of a GalNAc ligand on the 5′end of the sense strand outperforms linkage at the 3′ sense strand.

Example 7. Further Comparison of 3′ and 5′ Sense Strand Attachment Sitesfor GalNAc Targeting Ligands Using F12 Expression-Inhibiting OligomericCompounds in Wild Type Mice

To further assess the site of attachment of GalNAc ligands on the 3′ and5′ terminal ends of the sense strand of double-strandedexpression-inhibiting oligomeric compounds (double-stranded RNAiagents), compositions directed to the F12 gene were prepared having thesequences set forth in the following Table 3:

TABLE 3F12 expression-inhibiting oligomeric compounds (RNAi agent duplexes) ofExample 7. SEQ Duplex ID: AD02815 5′→3′ ID NO: Sense Strand Sequence:(NAG20)uauaugscsccaagaAfaGfugaaagacc(invdA) 6 (AM03640-SS)Antisense Strand Sequence: usGfsgucuuUfcAfcuuUfcuugggcsuscuAu 7(AM03157-AS) SEQ Duplex ID: AD02816 5′→3′ ID NO: Sense Strand Sequence:uAuAugscsccaagaAfaGfugaaagacca(NAG20) 8 (AM03641-SS)Antisense Strand Sequence: usGfsgucuuUfcAfcuuUfcuugggcsuscuAu 9(AM03157-AS)

In Table 3, above, the following notations are used:

(NAG20) has the chemical structure represented by Structure 4 herein.

Each strand of the F12 RNAi agents was synthesized according tophosphoramidite technology on solid phase used in oligonucleotidesynthesis using either a MerMade96E® (Bioautomation) or a MerMade12®(Bioautomation), and complementary strands were mixed by combiningequimolar RNA solutions (sense and antisense) in 0.2×PBS(Phosphate-Buffered Saline, 1×, Corning, Cellgro) to form the duplexes,following the methods generally described in Example 4 herein.

The F12 RNAi agents linked to the respective GalNAc ligand (i.e.,(NAG20)) were combined in a pharmaceutically acceptable buffer as knownin the art for subcutaneous (SC) injection.

The F12 RNAi agents linked to the respective GalNAc ligand weredelivered via SC injection. On day 1, SC injection was made into theloose skin on the back between the shoulders of 200 μl solution/20 gmouse containing either saline or a 3 mg/kg (mpk) dose of one of the twoRNAi agents (AD02815 or AD02816) in buffered saline. There were three(3) wild type mice per treatment group. As shown above in Table 3,AD02815 includes (NAG20) attached to the 5′ end of the sense strand,while AD02816 includes (NAG20) attached to the 3′ terminal end of thesense strand.

Serum samples from treated mice were taken on days 8, 15, 22 and 29 tomonitor knockdown. Knockdown was measured by quantifying circulatingmouse F12 protein (mF12) levels in serum by an internally developed mF12alphaLISA® (Perkin Elmer). Expression at a specific bleed date wasnormalized to the mean of the saline control group for that same date.

FIG. 13 shows the results from this experiment. At nadir (day 22),AD02816 showed approximately 60% reduction in circulating F12 proteinlevels, while AD02815 showed a 79% reduction. The data also show adifference in length of knockdown effect. At day 29, AD02816-treatedmice show 40% knockdown while AD02815-treated mice show 71% knockdownfrom saline levels. These data support linkage of a GalNAc ligand at the5′ terminal end of the sense strand.

Example 8. Lp(a) Expression-Inhibiting Oligomeric Compounds(Double-Stranded RNAi Agents) Linked to Targeting Ligands of Structure101 in Lp(a) Transgenic (Tg) Mice

Lp(a) expression-inhibiting oligomeric compounds (double-stranded Lp(a)RNAi agents) were prepared having the sequences set forth in thefollowing Table 5:

TABLE 4LP(a) expression-inhibiting oligomeric compounds (RNAi agent duplexes) ofExample 8. SEQ Duplex ID: AD03547 5′→3′ ID NO: Sense Strand(NAG29)uauauaasuuaucgaGfGfcucauucucsa(invAb) 10 Sequence: (AM04498-SS)Antisense Strand usGfsasGfaAfuGfaGfccuCfgAfuAfausuAUAUA 11Sequence: (AM04507-AS) SEQ Duplex ID: AD03549 5′→3′ ID NO: Sense Strand(NAG25)uauauaasuuaucgaGfGfcucauucucsa(invAb) 12 Sequence: (AM04502-SS)Antisense Strand usGfsasGfaAfuGfaGfccuCfgAfuAfausuAUAUA 13Sequence: (AM04507-AS)

In Table 4, above, the following notations are used:

(NAG25) has the chemical structure represented by Structure 101 herein.

Each strand of the Lp(a) RNAi agents was synthesized according tophosphoramidite technology on solid phase used in oligonucleotidesynthesis using either a MerMade96E® (Bioautomation) or a MerMade12®(Bioautomation), and complementary strands were mixed by combiningequimolar RNA solutions (sense and antisense) in 0.2×PBS(Phosphate-Buffered Saline, 1×, Corning, Cellgro) to form the duplexes,following the methods generally described in Example 4 herein.

Lp(a) transgenic (Tg) mice (Frazer K A et al 1995, Nature Genetics9:424-431) were used to evaluate the efficacy of double-stranded RNAiagents with conjugated N-acetyl-galactosamine ligands in vivo. Thismouse expresses human apo(a) from a YAC containing the full LPA gene(encoding apo(a) protein) with additional sequences both 5′ and 3′, aswell as the human apoB-100, thereby producing humanized Lp(a) particles(hereinafter referred to as “Lp(a) Tg mice.”) (Callow M J et al 1994,PNAS 91:2130-2134).

The Lp(a) RNAi agents linked to the respective GalNAc ligands (i.e.,(NAG25) or (NAG29)) were combined in a pharmaceutically acceptablebuffer as known in the art for subcutaneous (SC) injection.

The Lp(a) RNAi agents linked to the respective GalNAc ligands (i.e.,(NAG25) or (NAG29)) at the 5′ end of the sense strand were delivered viaSC injection. On day 1, SC injection was made into the loose skin on theback between the shoulders of 200 μl solution/20 g mouse containingeither saline or a 1 mg/kg (mpk) dose of the respective Lp(a) RNAi agent(AD03547 or AD03549) in buffered saline. There were four (4) Lp(a) Tgmice per treatment group.

Serum samples from treated mice were taken on days −1 (pre-dose), 5, 11,16, 22, 29, and 36. Knockdown was determined by calculating circulatingLp(a) particle levels in serum. Lp(a) particle levels were measured on aCobas® Integra 400 (Roche Diagnostics) according to the manufacturer'srecommendations. For normalization, Lp(a) level for each animal at atime point was divided by the pre-dose level of expression in thatanimal (in this case at day −1) to determine the ratio of expression“normalized to day −1.” Expression at a specific time point was thennormalized to the saline control group by dividing the “normalized today −1” ratio for an individual animal by the mean “normalized to day−1” ratio of all mice in the saline control group. This resulted inexpression for each time point normalized to that in the control group.Experimental error is given as standard deviation.

Results are shown in FIG. 14. AD03549 (NAG25) showed 71% knockdown atnadir (day 16), and AD03547 (NAG29) showed 81% knockdown at nadir (day11). Both triggers showed similar recovery curves after nadir, with lessthan 26% knockdown on day 36. These data support that the GalNAc ligandsshown are comparable in both initial knockdown activity and duration ofknockdown in Lp(a) Tg mice with a single 1 mg/kg dose.

Example 9. Lp(a) Knockdown in Lp(a) Transgenic (Tg) Mice FollowingAdministration of Lp(a) Expression-Inhibiting Oligomeric Compounds(Double-Stranded RNAi Agents) Linked to Targeting Ligand Structure 101

Lp(a) expression-inhibiting oligomeric compounds (double-stranded Lp(a)RNAi agents) were prepared having the sequences set forth in thefollowing Table 5:

TABLE 5LP(a) expression-inhibiting oligomeric compounds (RNAi agent duplexes) ofExample 9. SEQ Duplex ID: AD03272 5′→3′ ID NO: Sense Strand(NAG25)uauausasguuaucgAfGfGfcucauucuc(invdA) 14 Sequence: (AM04138-SS)Antisense Strand usGfsaGfaAfuGfaGfccuCfgAfuAfaCfucsusuAu 15 Sequence:(AM02860-AS)

In Table 5, (NAG25) is the same structure as shown in Example 8, above,and has the chemical structure represented by Structure 101 herein.

Each strand of the Lp(a) RNAi agents was synthesized according tophosphoramidite technology on solid phase used in oligonucleotidesynthesis using either a MerMade96E® (Bioautomation) or a MerMade12R®(Bioautomation), and complementary strands were mixed by combiningequimolar RNA solutions (sense and antisense) in 0.2×PBS(Phosphate-Buffered Saline, 1×, Corning, Cellgro) to form the duplexes,following the methods generally described in Example 4 herein.

Lp(a) Tg mice were used to evaluate the efficacy of double-stranded RNAiagents with conjugated N-acetyl-galactosamine ligands in vivo.

The Lp(a) RNAi agent linked to targeting ligand Structure 101 wascombined in a pharmaceutically acceptable buffer as known in the art forsubcutaneous (SC) injection.

The Lp(a) RNAi agent linked to the targeting ligand at the 5′ end of thesense strand was delivered via SC injection. On day 1, a SC injectionwas administered into the loose skin on the back between the shouldersat 200 μl solution/20 g mouse of either saline or a 1 mg/kg (mpk) doseof RNAi agent AD03272 in buffered saline. There were four (4) Lp(a) Tgmice per treatment group.

Serum samples from treated mice were taken on days −1 (pre-dose), 8, 15,22, 29, 36, and 43. Knockdown was determined by calculating circulatingLp(a) particle levels in serum. Lp(a) particle levels were measured on aCobas® Integra 400 (Roche Diagnostics) according to the manufacturer'srecommendations. For normalization, Lp(a) level for each animal at atime point was divided by the pre-dose level of expression in thatanimal (in this case at day −1) to determine the ratio of expression“normalized to day −1.” Expression at a specific time point was thennormalized to the saline control group by dividing the “normalized today −1” ratio for an individual animal by the mean “normalized to day−1” ratio of all mice in the saline control group. This resulted inexpression for each time point normalized to that in the control group.Experimental error is given as standard deviation.

Results are shown in FIG. 15. AD03272 showed 88% knockdown at nadir (day15), and maintained knockdown of 75% at day 29. These data support thatthe targeting ligand of Structure 1008 can target LPA-targeted RNAiagents to the liver and obtain >85% knockdown with a single 1 mg/kg dosein transgenic mice.

Example 10. Apolipoprotein(a) (Apo(a)) Knockdown in Apo(a) Transgenic(Tg) Mice Following Administration of Lp(a) Expression-InhibitingOligomeric Compounds (Double-Stranded RNAi Agents) Linked to TargetingLigand Structures 101, 102, and 103

Lp(a) expression-inhibiting oligomeric compounds (double-stranded Lp(a)RNAi agents) were prepared having the sequences set forth in thefollowing Table 4:

TABLE 6LP(a) expression-inhibiting oligomeric compounds (RNAi agent duplexes) ofExample 10. SEQ Duplex ID: AD03275 5′→3′ ID NO: Sense Strand Sequence:(NAG25)uauausasguuaucgAfGfGfcucauucuc(invdA) 16 (AM04138-SS) AntisenseusGfsagaauGfaGfccuCfgauaacucsusuau 17 Strand Sequence: (AM04133-AS) SEQDuplex ID: AD03341 5′→3′ ID NO: Sense Strand Sequence:(NAG26)uauausasguuaucgAfGfGfcucauucuCM(invdA) 18 (AM04233-SS) AntisenseusGfsagaauGfaGfccuCfgauaacucsusuau 19 Strand Sequence: (AM04133-AS) SEQDuplex ID: AD03421 5′→3′ ID NO: Sense Strand Sequence:(NAG27)uauausasguuaucgAfGfGfcucauucuCM(invdA) 20 (AM04372-SS) AntisenseusGfsagaauGfaGfccuCfgauaacucsusuau 21 Strand Sequence: (AM04133-AS)

In Table 6, above, the following notations are used:

Additionally, (NAG25) is the same structure as shown in Example 8,above, and has the chemical structure represented by Structure 101herein. (NAG26) has the chemical structure represented by Structure 102herein. (NAG27) has the chemical structure represented by Structure 103herein. As shown above in Table 7, other than the different targetingligand selected, the compositions are identical.

Each strand of the Lp(a) RNAi agents was synthesized according tophosphoramidite technology on solid phase used in oligonucleotidesynthesis using either a MerMade96E® (Bioautomation) or a MerMade12®(Bioautomation), and complementary strands were mixed by combiningequimolar RNA solutions (sense and antisense) in 0.2×PBS(Phosphate-Buffered Saline, 1×, Corning, Cellgro) to form the duplexes,following the methods generally described in Example 10 herein.

Apo(a) transgenic (Tg) mice were used to evaluate the efficacy ofdouble-stranded RNAi agents with conjugated N-acetyl-galactosamineligands in vivo. Apo(a) Tg mice (Frazer K A et al 1995, Nature Genetics9:424-431) express human apo(a) from a YAC containing the full LPA gene(encoding apo(a) protein) with additional sequences both 5′ and 3′(hereinafter referred to as “apo(a) Tg mice”).

The Lp(a) RNAi agents linked to the respective GalNAc ligands (i.e.,(NAG25), (NAG26), or (NAG27)) were combined in a pharmaceuticallyacceptable buffer as known in the art for subcutaneous (SC) injection.

The Lp(a) RNAi agents linked to the respective GalNAc ligands (i.e.,(NAG25), (NAG26), or (NAG27)) at the 5′ end of the sense strand weredelivered via SC injection. On day 1, a SC injection was administeredinto the loose skin on the back between the shoulders of 200 μlsolution/20 g mouse containing either saline or a 1 mg/kg (mpk) dose ofthe respective RNAi agent (AD03275, AD03341, or AD03421) in bufferedsaline. There were three (3) apo(a) Tg mice per treatment group.

Serum samples from treated mice were taken on days −1 (pre-dose), 8, 15,22, 29, 36 and 43. Knockdown was determined by monitoring circulatingapo(a) protein levels in serum using an ELISA for apo(a) (Abcam). Fornormalization, apo(a) level for each animal at a time point was dividedby the pre-treatment level of expression in that animal (in this case atday −1) to determine the ratio of expression “normalized to day −1”.Expression at a specific time point was then normalized to the salinecontrol group by dividing the “normalized to day −1” ratio for anindividual animal by the mean “normalized to day −1” ratio of all micein the saline control group. This resulted in expression for each timepoint normalized to that in the control group. Experimental error isgiven as standard error of the mean.

Results are shown in FIG. 16. Lp(a) RNAi agent AD03275, containingtargeting ligand Structure 101 (NAG25), showed 82% knockdown at nadir(day 22), and maintained knockdown of 72% at day 29. Lp(a) RNAi agentAD03341, containing targeting ligand Structure 102 (NAG26), showed 87%knockdown at nadir (day 15), however, knockdown at day 29 was 45%,indicating an increased return to pre-dose apo(a) levels. Lp(a) RNAiagent AD03421, containing targeting ligand Structure 103 (NAG27), showed70% knockdown at nadir (day 15), and knockdown at day 29 was 50%. Thesedata support that Structure 101 (NAG25), Structure 102 (NAG26), andStructure 103 (NAG27), all initially show similar knockdown activity.However, these data also show that AD03275 (Structure 101 (NAG25)) hassuperior duration and maintains knockdown (72% knockdown on day 29)better than Structure 102 (NAG26) and Structure 103 (NAG27).

Example 11. LP(a) Expression-Inhibiting Oligomeric Compounds(Double-Stranded RNAi Agents) Linked to Targeting Ligand Structure 101in Cynomolgus Monkeys

Five different LPA RNAi agents linked to targeting ligand represented bystructure 101 were prepared to evaluate their performance in cynomolgusmacaque (Macaca fascicularis) primates: AD03460, AD03536, AD03851,AD03853, and AD04110.

Each strand of the Lp(a) RNAi agents was synthesized according tophosphoramidite technology on solid phase used in oligonucleotidesynthesis using either a MerMade96E® (Bioautomation) or a MerMade12®(Bioautomation), and complementary strands were mixed by combiningequimolar RNA solutions (sense and antisense) in 0.2×PBS(Phosphate-Buffered Saline, 1×, Corning, Cellgro) to form the duplexes,following the methods generally described in Example 4 herein.

The targeting ligands for all five (5) Lp(a) RNAi agents were added tothe 5′ terminal end of the sense strand, using non-nucleosidephosphoramidite synthesis generally described herein and known in theart. The targeting ligand for each of the Lp(a) RNAi agents was linkedto the 5′ terminal end of the respective RNAi agent using the followingphosphoramidite compound:

AD03460 and AD03536 included targeting ligand (NAG25) conjugated to the5′ end of the sense strand of the respective RNAi agent. (NAG25) has thesame structure as shown in Example 8, above.

AD03851, AD03853, and AD04110 contained targeting ligand (NAG25)sconjugated to the 5′ end of the sense strand of the respective RNAiagent.

Blood samples were drawn and analyzed for Lipoprotein(a) levels on day 8and day 15. Lp(a) levels were normalized to average of three pre-dosevalues. Normalized Lp(a) levels are reported in the following table:

Normalized Lp (a) Normalized Lp (a) Day 8 Day 15 Saline 1.01 ± 0.06 1.15± 0.07 AD03460 0.68 ± 0.12 0.40 ± 0.13 AD03536 0.54 ± 0.07 0.21 ± 0.06AD03851 0.41 ± 0.08 0.18 ± 0.08 AD03853 0.50 ± 0.23 0.27 ± 0.17 AD041100.59 ± 0.13 0.43 ± 0.10

These data show that significant knockdown was achieved in cynomolgusmonkeys at 2 mg/kg (mpk) doses of multiple different Lp(a) RNAi agentsconjugated to the same targeting ligand structure of Structure 101herein.

Example 12: F12 Expression-Inhibiting Oligomeric Compounds(Double-Stranded RNAi Agents) Linked to Targeting Ligands of Structure101 in Cynomolgus Monkeys

F12 expression-inhibiting oligomeric compounds (double-stranded F12 RNAiagents) were prepared having the sequences set forth in the followingTable 7:

TABLE 7F12 expression-inhibiting oligomeric compounds (RNAi agent duplexes) ofExample 12. SEQ Duplex ID: AD03635 5′→3′ ID NO: Sense Strand Sequence:(NAG25)uauaugscsccaagaAfaGfugaaagacc(invdA) 22 (AM04130-SS) AntisenseusGfsgucuuUfcAfcuuUfcuugggcsuscuAu 23 Strand Sequence: (AM03157-AS)

In Table 7, above, (NAG25) represents the same structure as shown inExample 8, above, and is represented by Structure 101 herein.

Each strand of the Lp(a) RNAi agents was synthesized according tophosphoramidite technology on solid phase used in oligonucleotidesynthesis using either a MerMade96E® (Bioautomation) or a MerMade12®(Bioautomation), and complementary strands were mixed by combiningequimolar RNA solutions (sense and antisense) in 0.2×PBS(Phosphate-Buffered Saline, 1×, Corning, Cellgro) to form the duplexes,following the methods generally described in Example 4 herein.

The F12 RNAi agent conjugated to the targeting ligand at the 5′ end ofthe sense strand was made and combined in a pharmaceutically acceptablebuffer as known in the art for subcutaneous (SC) injection.

On day 1, cynomolgus macaque (Macaca fascicularis) primates wereinjected subcutaneously with 3 mg/kg of AD03635. Three (3) monkeys weredosed per treatment group.

Serum samples from treated cynomolgus monkeys were taken on day −7 andday 1 (pre-dose), and on days 8, 15 and 22 to monitor knockdown.Knockdown was measured by quantifying circulating cyno F12 protein(cF12) levels in serum by a human F12 ELISA kit (Molecular Innovations).cF12 levels for each animal at a respective time point was divided bythe pre-treatment level (average of day −7 and day 1) of expression inthat animal to determine the ratio of expression “normalized topre-dose”. Experimental error is given as standard deviation.

FIG. 17 shows the results. The F12 RNAi agent linked to (NAG25)(Structure 101 herein) showed knockdown in cynomolgus monkeys.

Example 13: Alpha-1 Antitrypsin Expression-Inhibiting OligomericCompounds (Double-Stranded RNAi Agents) Linked to Targeting Ligands ofStructure 101 in PiZ Transgenic Mice

To evaluate RNAi agents directed to the alpha-1 antitrypsin (AAT) genein vivo, a transgenic PiZ mouse model (PiZ mice) was used. PiZ miceharbor the human PiZ AAT mutant allele and model human AATD (Carlson etal., Journal of Clinical Investigation 1989).

AAT expression-inhibiting oligomeric compounds (double stranded RNAiagents) were prepared having the sequences set forth in the followingTable 8:

TABLE 8AAT expression-inhibiting oligomeric compounds (RNAi agent duplexes) ofExample 13. SEQ Duplex ID: AD04454 5′→3′ ID NO: Sense(NAG25)scsgauaucaUfCfAfccaaguuccsa(invAb) 24 Strand Sequence:(AM05662-SS) Antisense Strand usGfsgAfaCfuugguGfaUfgAfuAfusCfsg 25Sequence: (AM05663-AS)

In Table 8, (NAG25)s has the chemical structure as shown in Example 11,above.

The AAT RNAi agent was prepared in a pharmaceutically acceptable salinebuffer and administered by subcutaneous (SC) injection into the looseskin on the back between the shoulders of 200 μl solution/20 g mouse toPiZ mice to evaluate knockdown of AAT gene expression. Each mousereceived a single SC dose of 5 mg/kg (mpk) of AD04454. Three mice weredosed with the AAT RNAi agent (n=3).

Plasma samples were drawn and analyzed for AAT (Z-AAT) protein levels ondays −1, day 1 (pre-dose), day 8, and day 15. AAT levels were normalizedto day 1 (pre-dose) AAT plasma levels. Protein levels were measured byquantifying circulating human Z-AAT levels in plasma by an ELISA kit.

The average normalized AAT (Z-AAT) levels are shown in FIG. 18. The AATRNAi agent linked to the targeting ligand of Structure 101 herein showedknockdown in PiZ transgenic mice.

Example 14: F12 Knockdown in Wild Type Mice Following Administration ofF12 Expression-Inhibiting Oligomeric Compounds (Double-Stranded RNAiAgents) Linked to Targeting Ligands of Structure 101

F12 expression-inhibiting oligomeric compounds (double-stranded F12 RNAiagents) were prepared having the sequences set forth in the followingTable 9:

TABLE 9F12 expression-inhibiting oligomeric compounds (RNAi agent duplexes) ofExample 14. SEQ Duplex ID: AD03632 5′→3′ ID NO: Sense(NAG25)gcgaugscsccaagaAfaGfugaaagacc(invdA) 26 Strand Sequence:(AM04613-SS) Antisense Strand usGfsgucuuUfcAfcuuUfcuugggcsasucgc 27Sequence: (AM04048-AS)

In Table 9, (NAG25) has the chemical structure as shown in Example 8,above, and is 5 represented by Structure 101 disclosed herein.

Each strand of the F12 RNAi agents was synthesized according tophosphoramidite technology on solid phase used in oligonucleotidesynthesis using either a MerMade96E® (Bioautomation) or a MerMade12®(Bioautomation), and complementary strands were mixed by combiningequimolar RNA solutions (sense and antisense) in 0.2×PBS(Phosphate-Buffered Saline, 1×, Corning, Cellgro) to form the duplexes,following the methods generally described in Example 10 herein.

The F12 RNAi agents conjugated to the respective GalNAc targeting ligand(i.e., (NAG25)) was combined in a pharmaceutically acceptable buffer asknown in the art for subcutaneous (SC) injection.

The composition was delivered via SC injection. On day 1, a SC injectionwas administered into the loose skin on the back between the shouldersof 200 ul solution/20 g mouse containing either saline or a 3 mg/kg(mpk) dose of AD03632 in buffered saline. There were three (3) wild typemice per treatment group. As shown above, AD03632 includes the structure(NAG25) linked at the 5′ terminal end of the sense strand.

Serum samples from treated mice were taken on days −1 (pre-dose), 8, 1522, 29 and 36 to monitor knockdown. Knockdown was measured byquantifying circulating mouse F12 protein (mF12) levels in serum by aninternally developed mF12 alphaLISA® (Perkin Elmer). mF12 levels foreach animal at a respective time point was divided by the pre-treatmentlevel of expression in that animal to determine the ratio of expression“normalized to pre-dose”. Expression at a specific time point was thennormalized to the saline control group by dividing the “normalized today pre-dose” ratio for an individual animal by the mean “normalized today pre-dose” ratio of all mice in the saline control group. Thisresulted in expression for each time point normalized to that in thecontrol group. Experimental error is given as standard deviation.

Results from this study are shown in FIG. 19. AD03632, which includestargeting ligand Structure 101 disclosed herein attached at the 5′terminal end, shows significant knockdown across all time points.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A targeting ligand selected from the group consisting of:

wherein n is an integer from 1 to 20 and Targeting Moiety is selected from the group consisting of: carbohydrates, glycans, haptens, vitamins, folate, biotin, aptamers, peptides, insulin, EGF and transferrin.
 2. The targeting ligand of claim 1, wherein the Targeting Moiety is a carbohydrate.
 3. The targeting ligand of claim 1, wherein the targeting ligand is linked to an expression-inhibiting oligomeric compound.
 4. The targeting ligand of claim 3, wherein the expression-inhibiting oligomeric compound is an RNAi agent.
 5. The targeting ligand of claim 4, wherein the RNAi agent is double-stranded.
 6. The targeting ligand of claim 4, wherein the RNAi agent comprises one or more modified nucleotides.
 7. The targeting ligand of claim 4, wherein the targeting ligand is linked at the 3′ or 5′ terminal end of the RNAi agent.
 8. The targeting ligand of claim 5, wherein the double-stranded RNAi agent is linked to the targeting ligand at the 5′ terminal end of the sense strand of the RNAi agent.
 9. The targeting ligand of claim 4, wherein the RNAi agent is linked to the targeting ligand via a phosphate group, phosphorothioate group, or a phosphonate group.
 10. The targeting ligand of claim 1, wherein the targeting ligand comprises the structure:

wherein n is an integer from 1 to
 20. 11. The targeting ligand of claim 10, wherein n is
 6. 12. The targeting ligand of claim 1, wherein the targeting ligand is selected from the group consisting of:


13. A method of inhibiting expression of a target nucleic acid in a cell, the method comprising administering to the cell an effective amount of an expression-inhibiting oligomeric compound conjugated to the targeting ligand of claim
 1. 14. The method of claim 13, wherein the cell is present in a subject and the effective amount is a therapeutically effective amount.
 15. The method of claim 14, wherein the subject is a human.
 16. The method of claim 13, wherein the expression-inhibiting oligomeric compound is an RNAi agent.
 17. A method of treating a disease or disorder that would benefit from administration of an expression-inhibiting oligomeric compound, the method comprising administering a therapeutic amount of the targeting ligand of claim 1 linked to an expression-inhibiting oligomeric compound to a subject in need thereof.
 18. The method of claim 17, wherein the expression-inhibiting oligomeric compound is an RNAi agent.
 19. A composition comprising the targeting ligand of claim 3 and a pharmaceutically acceptable excipient.
 20. A method of treating a disease or disorder that would benefit from administration of an expression-inhibiting oligomeric compound, the method comprising administering a therapeutic amount of the composition of claim 19 to a subject in need of treatment therefor. 